Florida panthers are endangered pumas that currently persist in reduced patches of habitat in South Florida, U.S. We performed mitogenome reference-based assemblies for most parental lines of the admixed Florida panthers that resulted from the introduction of female Texas pumas into South Florida in 1995. With the addition of two puma mitogenomes, we characterized 174 single nucleotide polymorphisms (SNPs) across 12 individuals. We defined five haplotypes (Pco1−Pco5), one of which (Pco1) had a geographic origin exclusive to Costa Rica and Panama and was possibly introduced into the Everglades National Park, Florida, prior to 1995. Haplotype Pco2 was native to Florida. Haplotypes Pco3 and Pco4 were exclusive to Texas, whereas haplotype Pco5 had an undetermined geographic origin. Phylogenetic inference suggests that haplotypes Pco1−Pco4 diverged ~202,000 (95% HPDI = 83,000−345,000) years ago and that haplotypes Pco2−Pco4 diverged ~61,000 (95% HPDI = 9,000−127,000) years ago. These results are congruent with a south-to-north continental expansion and with a recent North American colonization by pumas. Furthermore, pumas may have migrated from Texas to Florida no earlier than ~44,000 (95% HPDI = 2,000−98,000) years ago. Synonymous mutations presented a greater mean substitution rate than other mitochondrial functional regions: nonsynonymous mutations, tRNAs, rRNAs, and control region. Similarly, all protein-coding genes were under predominant negative selection constraints. We directly and indirectly assessed the presence of potential deleterious SNPs in the ND2 and ND5 genes in Florida panthers prior to and as a consequence of the introduction of Texas pumas. Screenings for such variants are recommended in extant Florida panthers.