The apicomplexan haemoparasite Theileria equi, a causative agent of equine piroplasmosis, is an established pathogen of significant welfare and economic concern within the Croatian equine population. A previous large surveillance study of T. equi has identified two distinct parasite populations, one in the north and one in the south, geographically separated by the Dinaric Alps, which traverse the country. This study aimed to further investigate the genetic diversity within these two populations, focussing on allelic variability of the equi merozoite antigen gene, ema-1. Following nested PCR of DNA isolates, the generated ema-1 amplicons were subsequently sequenced and compared by phylogenetic analysis to available sequences representing previously described ema-1 genotypes (groups A–C). Isolates from the southern T. equi population clustered with the existing ema-1 groups A and B. Strikingly, isolates from the northern population clustered into two novel ema-1 genotypes, named g