Basidiobolus spp. are a significant causal agent of infections in man and animals including gastrointestinal basidiobolomycosis (GIB). Little information is available on how these infections are acquired or transmitted, apart from the postulation that environmental sources are implicated. This study aimed to identify Basidiobolus spp. from GIB patients and from the house gecko as a possible source of infection in Aseer, Saudi Arabia. Basidiobolus spp. were isolated from patient specimens (colonic mass biopsy) and from house gecko (gut contents) from Muhayil Aseer areas, in southern Saudi Arabia, using Sabouraud dextrose agar (SDA) which was incubated aerobically for up to three weeks at 30°C. Isolated fungi were initially identified using classical mycological tools and confirmed by sequence analysis of the large subunit ribosomal RNA gene. Cultured specimens from humans and geckos revealed phenotypically similar zygomycete-like fungi which conform to those of Basidiobolus species. The strains formed a monophyletic clade in the 28S ribosomal RNA gene phylogenetic tree. They shared 99.97% similarity with B. haptosporus and 99.97% with B. haptosporus var. minor but have a relatively remote similarity to B. ranarum (99.925%). One isolates from a gecko (L3) fall within the sub-clade encompassing B. haptosporus strain NRRL28635. The study strongly suggests a new and a serious causal agent of GIB related to Basidiobolus haptosporus. The isolation of identical Basidiobolus haptosporus-like strains from humans and lizards from one area is an important step towards identifying risk factors for GIB. Research is underway to screen more environmental niches and fully describe the Basidiobolus strains.