In 1995, eight female pumas from Texas (Puma concolor stanleyana) were introduced into five areas in South Florida to remediate morphological and biomedical correlates of inbreeding depression that threatened the long-term survival of Florida panthers (P. c. coryi). Initial analysis of the results of this genetic introgression initiative has highlighted its success, including growth of the population, increased genetic heterozygosity, and reduced frequencies of physical traits associated with inbreeding, including cryptorchidism. However, panthers remain isolated from other puma populations and natural gene flow is not currently plausible. Using data collected from 65 panthers in the post-genetic introgression era (1998–2020), we assessed the benefits accrued to male panther reproductive parameters via this management initiative, the effect of genetic variables, and compared results to historic data. Microsatellite data were used to determine individual heterozygosity levels and qualify panther genetic ancestry as admixed (i.e., panthers descended from crosses with female Texas puma) or canonical (i.e., panthers that comprised most of the population prior to genetic introgression). A total of 47 admixed and 10 canonical panthers were identified; mean individual heterozygosity of canonical panthers (0.39) was significantly different from admixed panthers (0.63; t₅₄ = 4.7, P-value < 0.001). Admixed panthers exhibited higher mean values for total testicular volume, structurally normal spermatozoa, and total sperm in comparison to canonical panthers. The progressive improvement of sperm metrics demonstrates that the benefits of genetic introgression continue to have a positive impact on the population >25 years since the release of the Texas pumas. However, the Florida panther population remains small, isolated, and vulnerable to deterministic and stochastic events that warrants continued monitoring to assess when additional genetic introgression initiatives will need to be implemented in the future.