Blastocystis sp. is a protozoan parasite, commonly found in the gastrointestinal tracts of animals and humans globally. The parasitic species has wide genetic diversity. Currently the mammalian and avian isolates of the parasite are grouped into 17 well known subtypes (STs), of which ten (ST1-ST9, ST12) are reported in humans. To assess the genetic diversity of Blastocystis sp. in wildlife, a total of 200 fresh fecal samples were collected from 32 mammalian wildlife species in Bangladesh National Zoo. Blastocystis sp. was screened and subtyped by PCR amplification and sequencing of the small subunit ribosomal RNA (SSU rRNA) gene. The minimum prevalence of Blastocystis sp. infection was 15.5% (31/200) in zoo animals. Eight out of 32 wildlife animal species (25.0%) were infected with Blastocystis sp. Among them, the occurrence of Blastocystis sp. was higher in non-human primates (NHPs) (31.8%) than that in herbivores (4.9%) and carnivores (0). Nucleotide sequence analysis of the SSU rRNA gene revealed seven different Blastocystis sp. subtypes, such as ST1, ST2, ST3, ST10, ST11, ST13 and ST14 in the wild animals. ST3 was the dominant subtype (41.9%, 13/31) being detected in NHPs. Of the 31 Blastocystis sp. isolates from the wild animals, 24 (77.4%) isolates belonged to the most common subtypes (ST1 to ST3) found in humans. This is the first molecular study of Blastocystis sp. in wild animals in Bangladesh. This study highlights the remarkable genetic diversity in Blastocystis sp. isolates from zoo animals and provides the first molecular evidence from spotted deer, gayal and grey langur. Due to circulation of large percentage of potentially zoonotic subtypes in the wild animals, there is a higher risk of zoonotic transmission of Blastocystis sp. in the zoo keepers and visitors.