Blastocystis is a common intestinal protist with global distribution, frequently found in humans and various animals. Despite its prevalence, its role in human health remains debated, oscillating between being a harmless commensal and a potential pathogen, also linked to gastrointestinal disorders, such as irritable bowel syn-drome (IBS) and inflammatory bowel disease (IBD). In Italy, the genetic and spatial diversity of Blastocystis remains understudied, despite the country's diverse urbanized and environmental landscapes. This study focuses on the haplotypic and spatial diversity of clinical isolates of Blastocystis across two different Italian regions, with an emphasis on subtype distribution and genetic variation. Using a network-based haplotype analysis, the study reveals a heterogeneous subtype distribution, with subtype ST4 being the most prevalent, followed by ST3, ST1, and ST2. Notably, ST4 shows limited haplotypic variation, suggesting a more stable population structure potentially linked to a human-adapted lineage. In contrast, ST1 and ST2 exhibit greater genetic diversity, likely due to ongoing zoonotic transmission. These findings contribute to understanding the epidemiology of Blastocystis in Italy and highlight the need for further research on its pathogenic potential and transmission dynamics.