Purpose <p><i>Trypanosoma theileri</i> is an opportunistic parasite that has worldwide distribution. This parasite has been detected in various hosts including cattle. Normally <i>T. theileri</i> can cause mild infection but it may lead to disease among animals in case of mixed infection or immunosuppression. In the present study, it was aimed to investigate the presence, prevalence, and to detect genotypes of <i>T. theileri</i> in cattle from different provinces in Türkiye using molecular techniques.</p> Methods <p>In this study, 517 cattle blood collected from different provinces (Giresun, Samsun, Tokat, Sivas, Çorum, Trabzon, and Kastamonu) of Türkiye. Genomic DNA was extracted from blood samples using a commercial kit. The obtained gDNAs were screened for the presence of <i>T. theileri</i> using PCR. Partial parts of the <i>CATL</i> gene of randomly selected four positive samples were sequenced to determine the phylogenetic position of <i>T. theileri</i> Türkiye isolates.</p> Results <p>PCR results showed that 18 out of 517 samples (3.48%) were positive in terms of <i>T. theileri</i>. The highest prevalence of <i>T. theileri</i> was observed in animals over 3 years of age (3.98%), followed by animals in the 1–3-year age group (3.16%). The prevalence of <i>T. theileri</i> was higher in female animals (3.62%) than in male animals (2.91%). The phylogenetic analyses of positive samples showed that <i>T. theileri</i> Türkiye isolates were clustered with the Tth IIB genotype of the <i>T. theileri</i> TthII lineage.</p> Conclusion <p>In conclusion, the presence and prevalence of <i>T. theileri</i> in cattle were determined by molecular analyses for the first time in Türkiye. Moreover, Tth IIB genotype was also reported in Türkiye with this work. Although <i>T. theileri</i> is not thought to cause serious clinical symptoms in hosts, it should not be overlooked that it can cause significant economic losses in mixed infections with different pathogens.</p>