Tissue specimens from four species of Neotropical small cats (Oncifelis geoffroyi, N = 38; O. guigna, N = 6; Leopardus tigrinus, N = 32; Lynchailurus colocolo, N = 22) collected from throughout their distribution were examined for patterns of DNA sequence variation using three mitochondrial genes, 16S rRNA, ATP8, and NADH-5. Patterns between and among O. guigna and O. geoffroyi individuals were assessed further from size variation at 20 microsatellite loci. Phylogenetic analyses using mitochondrial DNA sequences revealed monophyletic clustering of the four species, plus evidence of natural hybridization between L. tigrinus and L. colocolo in areas of range overlap and discrete population subdivisions reflecting geographical isolation. Several commonly accepted subspecies partitions were affirmed for L. colocolo, but not for O. geoffroyi. The lack of geographical substructure in O. geoffroyi was recapitulated with the microsatellite data, as was the monophyletic clustering of O. guigna and O. geoffroyi individuals. L. tigrinus forms two phylogeographic clusters which correspond to L.t. oncilla (from Costa Rica) and L.t. guttula (from Brazil) and which have mitochondrial DNA (mtDNA) genetic distance estimates comparable to interspecific values between other ocelot lineage species. Using feline-specific calibration rates for mitochondrial DNA mutation rates, we estimated that extant lineages of O. guigna diverged 0.4 million years ago (Ma), compared with 1.7 Ma for L. colocolo, 2.0 Ma for O. geoffroyi, and 3.7 Ma for L. tigrinus.