CSI: Crime Scene Investigation included LGBTQ+ characters and themes in various episodes, often in the context of crime investigation. While prejudice was typically portrayed negatively, the show's focus remained on solving cases rather than exploring queer identity in depth. Portrayals were largely incidental, neither consistently affirming nor overtly problematic, resulting in a neutral overall impact on LGBTQ+ representation.

CSI: Crime Scene Investigation has featured transsexual characters, notably in 'Identity Crisis' (S2E13). While attempting to address violence against trans individuals, the portrayal often utilized problematic tropes, framing trans identity as a source of shocking revelation, deception, and tragedy, with characters reacting negatively without sufficient narrative critique. The net impact is primarily negative due to these harmful depictions.

The show frequently depicts Christian institutions and individuals as hypocritical, fanatical, or as a backdrop for crime and moral failings. While individual characters may hold genuine faith, the narrative often uses Christian contexts to explore the darker aspects of human behavior, aligning with a skeptical, scientific viewpoint.