Blastocystis sp. is a common intestinal protozoan parasite that colonises humans and various animals, with rodents identified as significant reservoir hosts for zoonotic transmission. This study aimed to investigate the prevalence and subtype distribution of Blastocystis sp. among wild rats in Pulau Pinang, Malaysia, while evaluating the associations with host demographics and geographic factors. A total of 150 rodent faecal samples from 12 sampling sites across Pulau Pinang, Malaysia, were examined using microscopy, DNA extraction, and PCR amplification of the SSU-rRNA gene. The overall prevalence of Blastocystis was 25.3%, with Rattus norvegicus exhibiting the highest infection rate (34.5%), followed by R. rattus (19.4%), and Bandicota bengalensis (16.7%). No infections were reported in B. indica (0.0%). Statistical analysis revealed a significant association between Blastocystis infection and rodent species (c2 = 11.874, df = 3 and p = 0.008), whereas no association was indicated in gender, development stages, and sampling sites. Due to limited funding, sequencing was conducted on ten positive samples that exhibited well-defined bands. Out of ten, three zoonotic subtypes were identified namely, ST1 (allele 4), ST3 (allele 34), and ST4 (allele 94 and 133). The findings of this study emphasise the role of R. norvegicus as the major reservoir host in Pulau Pinang, Malaysia and highlight the zoonotic potential of Blastocystis in urban settings.