Domestic and wild felids are frequently parasitized by apicomplexan protozoa in the genus Cytauxzoon. Expanding species diversity has recently been described within this genus, with potential implications for epidemiology and pathogenesis. In light of these findings, this study assessed the genetic diversity of Cytauxzoon spp. in wild felids (n = 66) from different eco-regions of Brazil and Argentina. Of the 66 blood samples analyzed, 53 (80.3%) were 18S rRNA gene PCR-positive for Cytauxzoon spp., including 43 jaguars (Panthera onca) and 10 ocelots (Leopardus pardalis). Panthera onca specimens (100%, 43/43) were most frequently infected, followed by Leopardus pardalis (76.9%; 10/13). Cytauxzoon spp. were not detected in Leopardus braccatus (n = 1) or Puma concolor (n = 9). Phylogenetic analyses of fragments of the 18S rRNA, cytB, and cox-1 gene sequences from jaguars were closely related to Cytauxzoon felis. In contrast, sequences from ocelots were more closely associated with Cytauxzoon brasiliensis. Distance and haplotype analysis further confirmed the circulation of at least two distinct genovariants of C. felis among jaguars, as evidenced by their close positioning and low genetic divergence (0–0.14% for 18S rRNA, 0.37–0.56% for cytB, and 0.08–0.74% for cox-1). Additionally, sequence data from ocelots suggested that multiple genovariants of C. brasiliensis are circulating among these cats in different Brazilian eco-regions. Our study provides evidence of two distinct Cytauxzoon organisms parasitizing free-ranging and captive jaguars and ocelots, respectively, in Brazil and Argentina.