Molecular characterization of Sarcocystis fusiformis isolated from semi -arid region of India was done by subjecting the amplified products of 18S rDNA gene (approximately, 900bp) to gene sequencing and RFLP. Two S. fusiformis specific fragments of approximately 768bp and 99bp were seen in all the four PCR products after digestion with restriction enzyme (Dra1). Out of four, gene sequencing results were obtained successfully for one macroscopic, one microscopic and one pellet of bradyzoites which were then submitted in NCBI to receive accession number. Genes of Mathura isolates were aligned with published sequences of Sarcocystis spp. taking Neospora caninum and Theileria annulata as out-groups. Three isolates of Mathura showed more than 99% homology in nucleotide sequence among themselves. Gene sequencing and phylogenetic analysis showed Mathura isolates closer to S. fusiformis isolates of different geographic locations.