Tim Speedle was a trace and impressions expert with the Miami-Dade Crime Lab. Despite his skill as a CSI, Speed was habitually neglectful of proper gun maintenance, which contributed to his death in episode 301, "Lost Son," when his side arm jammed during a shootout. He died moments later in Horatio Caine's arms. He had been close friends with colleague Eric Delko, and had been the one to haze him when Eric first joined the lab (episode 308, "Speed Kills"). Even after Speedle's death, Delko remains protective of his memory and reputation (episodes 306, "Hell Night;" 323, "Whacked"). Speed's void on the team is filled by former patrol officer Ryan Wolfe.

Miami Dade CSI Trainee Natalia Boa Vista played by Eva LaRue, a new Brazilian Latina DNA analyst who upon first arriving, was only allowed to work on cold or unsolved cases due to the restrictions of her federal grant. She and Delko dated briefly. They broke it off after a pregnancy scare. She escaped an abusive marriage before joining the crew, when her association with a society for battered women was key to helping the team solve the murder of a woman (who had been killed by the victim's ex-husband) assuming an alias for protection. It was revealed at the end of the fourth season that she was the mole in the lab, but the negative information soiling the lab's reputation was not leaked by Boa Vista but by State Attorney Monica West, and Boa Vista had only reported information to the Feds that showed the lab in a positive light. In season 5, she, much to her distress, discovered her abusive ex-husband, Nick Townsend, was out of prison when he served her with a restraining order; he was working for a private company that cleans up crime scenes, which created a difficult situation with forcing her to make terms with him to get the restraining order dropped. The two shared a tense professional relationship until Nick's murder, a crime for which Boa Vista was briefly considered the prime suspect.

Miami Dade CSI Level 1 Detective Ryan Wolfe played by Jonathan Togo, Ryan was working as a police officer at the time he was hired by Horatio, who noted with approval that Ryan kept his firearm immaculately clean (his predecessor, Speedle, died in the line of duty as a direct result of his poor gun maintenance). He first appeared in the episode "Under the Influence", but did not become a regular cast member until "Hell Night". Ryan appears to believe in curses ("Curse of the Coffin"). Ryan was impaled in the eye with a nailgun in one episode ("Nailed"). Ryan was fired at the end of the episode "Burned" (5x22) for being directly linked to a murder suspect and lying about it. He did appear in the following episode as a crime scene expert and former CSI on TV. In that episode, he apologized to Horatio and was allowed back on the CSI team.

Miami Dade CSI Level 3 Detective Eric Delko played by Adam Rodriguez is a fingerprint and drug identification expert of Cuban and Russian descent. In episode 410, "Shattered", Delko's job is endangered when he is arrested for drug possession, but it turns out he had been buying the drugs for his sister Marisol, to ease the pain of her leukemia treatments. Delko's sister was murdered by a Mala Noche sniper after she married Delko's boss, Horatio Caine. He is also the team's underwater recovery expert. While trying to rescue a woman kidnapped by escapee Clavo Cruz, Delko was critically wounded by one of Cruz's henchmen. He survived, and is now back on the team.

Miami Dade Medical Examiner Dr. Alexx Woods played by Khandi Alexander, is a Miami-Dade medical examiner. Alexx began her medical career in New York as a physician and became a medical examiner with the CSI team after moving to Miami for personal reasons. She often talks to the dead bodies as she examines them, usually as a way of giving them comfort in death. She is married with a young son and daughter. Alexx is not the chief medical examiner, as proved in one the episodes when she was offered to transfer to the night shift by the Chief Medical Examiner, but rejected.

MDPD Homicide Detective Francis 'Frank' Tripp played by Rex Linn, is a Texan homicide detective who regularly accompanies the team to the crime scenes. He, having much knowledge of forensic procedures, has an appreciation for the capabilities of the CSI's and moreso of their results. He has a good working relationship with all the CSIs, particularly Calleigh. He is divorced, with three kids (episode 118, "Dispo Day"). Made regular in season 5, recurring previously.