Purpose <p><i>Enterocytozoon bieneusi</i> is recognized as the most common microsporidian parasite, exhibiting a broad host spectrum that includes a wide range of animal species, as well as humans. However, no comprehensive data are available on the prevalence, age-related distribution, or genotype diversity of <i>E. bieneusi</i> in preweaned calves in Türkiye. Therefore, this study aimed to provide comprehensive data and expand current knowledge on <i>E. bieneusi</i> in preweaned calves from different age groups.</p> Methods <p>We screened a total of 240 fecal samples collected from preweaned calves using nested PCR to determine the occurrence and age-related distribution of <i>E. bieneusi</i>. Genotyping of the detected isolates and evaluation of their zoonotic potential were subsequently performed based on ITS sequence analysis.</p> Results <p><i>Enterocytozoon bieneusi</i> was identified in 38 out of 240 fecal specimens, corresponding to an overall prevalence of 15.8%. Sequence analyses revealed the presence of two known genotypes, BEB8 and J. While genotype J was identified as the predominant genotype circulating among calves in the study area, genotype BEB8 was detected for the first time in a mammalian host in Türkiye. Phylogenetic analysis further revealed that both genotypes clustered within Group 2, which includes ruminant-adapted genotypes.</p> Conclusion <p>This is the first comprehensive report on the occurrence, age-related distribution, and genotyping of <i>E. bieneusi</i> in preweaned calves in Türkiye, highlighting the potential role of calves as reservoirs or carriers of this pathogen.</p>