Human immunodeficiency virus type 1 (HIV-1) in the male genital tract may comprise virus produced locally in addition to virus transported from the circulation. Virus produced in the male genital tract may be genetically distinct, due to tissue-specific cellular characteristics and immunological pressures. HIV-1 env sequences derived from paired blood and semen samples from the Los Alamos HIV Sequence Database were analyzed to ascertain a male genital tract-specific viral signature. Machine learning algorithms could predict seminal tropism based on env sequences with accuracies exceeding 90%, suggesting that a strong genetic signature does exist for virus replicating in the male genital tract. Additionally, semen-derived viral populations exhibited constrained diversity (P < 0.05), decreased levels of positive selection (P < 0.025), decreased CXCR4 coreceptor utilization, and altered glycosylation patterns. Our analysis suggests that the male genital tract represents a distinct selective environment that contributes to the apparent genetic bottlenecks associated with the sexual transmission of HIV-1.