FBI: Most Wanted features a prominent lesbian main character, Sheryll Barnes, whose identity and family life are consistently depicted with dignity and normalcy. The show integrates her queer family into the narrative without sensationalism, presenting a positive and affirming portrayal of LGBTQ+ lives and relationships within a mainstream procedural.

The show features an episode where a trans woman is the victim of a hate crime. The narrative focuses on the FBI's empathetic investigation to bring the perpetrators to justice, condemning anti-trans prejudice and affirming the worth of the victim's life despite the tragic outcome.

The show features female FBI agents, Sheryll Barnes and Kristin Gaines, who frequently engage in and win physical altercations against male suspects. These victories involve hand-to-hand combat, takedowns, and subduing opponents during arrests, without relying solely on firearms.

The show generally portrays Christian characters and communities with respect, often depicting their faith as a source of personal strength or moral guidance, and condemns any criminal misuse of religious tenets.

The series portrays Muslim characters and communities with nuance and respect, often distinguishing between individual extremist actions and the broader faith, and condemning Islamophobia or bigotry.