Trypanosomes species, including Trypanosoma evansi (T. evansi) and Trypanosoma theileri (T. theileri) have been reported in a wide range of mammalian species worldwide. We report the first genetic evidence of T. theileri in indigenous cattle originating from the Southern Punjab province of Pakistan. The archived blood samples (n=30) were taken from cattle clinically suggestive of Trypanosomiasis, screened through microscopic examination, and processed for amplifying hypervariable region in the 18S rDNA and sequence analysis. Only T. evansi and T. theileri infections were identified, where the presence of T. evansi (22/30, 73.33%) was higher than that of T. theileri (8/30, 26.6%). Single nucleotide polymorphism (SNPs) suggested considerable interspecies specific variations between T. theileri and T. evansi haplotypes than intraspecies where a few variations within T. theileri imply circulation of closely related haplotype in the cattle. The genetic variations among haplotypes of T. theileri and T. evansi coupled with inferred monophyletic clustering (bootstrap CI>90) suggest both as two distinct species. Future studies are required to screen a large population of susceptible hosts further and elucidate population dynamics of trypanosome species that can infect hosts either alone or as a co-infection.