Introduction: Cryptococcosis is a fungal infection predominantly caused by Cryptococcus neoformans. The prevalent genotype VNI of C. neoformans has a two-fold higher risk of fatality in cryptococcosis coinfected with HIV patients. Objective: We have isolated 49 C. neoformans from cryptococcosis patents during Apr 2010 to Jun 2015 from different samples. About 26 of these isolates were molecular typed to identify the prevalent molecular types from Western Maharashtra, India. Methods: Out of 47 clinical cryptococcal isolates from suspected cryptococcosis 26 were further subjected to genotyping by Multilocus sequence typing (MLST) method using 8 different house-keeping loci. All 28 DNA sequences were deposited in Genbank under the following accession numbers MF580727 to MF580754. Results: The phylogenetic tree was made to determine the relatedness between the isolates. About 26 C. neoformans var grubii isolates were divided into two genotypes VNI (%) and VNII (%). The 2 allele numbers for CAP59, SOD1, and URA5, 3 for LAC1 and PLB1 and 4 for GPD1 and IGS1 per gene loci were noted. Based on these allelic polymorphism genotypes were sub-typed into seven sequence types including three novel Sequence types (STs) from the Western part of India. Conclusions: Intra-species variation was observed among the isolates from this region of India. The molecular epidemiology from the current study suggests that there is a need to look for an environmental source for these novel STs.