Venom hyaluronidase is an enzyme belonging to the glycoside hydrolase family that plays a critical role in systemic envenomation by spreading toxins in tissue and destruction of the extracellular matrix. The roles of venom hyaluronidase during envenomation have been widely studied, but the identification of its variants and isoforms is still under investigation. In this study, we developed a filtering method to identify the exon-intron pattern, alternative splicing events and isoforms of hyaluronidase in A. crassicauda and H. lepturus scorpions using the RNA-seq technique. Furthermore, in silico anal. was performed to identify and characterize the hyaluronidases. The most important findings were that the scorpion hyaluronidase gene contains 5 exons, 4 introns and undergo to alternative splicing events. In A. crassicauda and H. lepturus datasets, a sequence denominated AcHase-1 and three denominated HLHase1, HLHase2 and HLHase3 were identified as hyaluronidase variants resp., which were found to have multiple isoforms that differed in the coding, non-coding or untranslated regions. The results showed that exon skipping, intron retention and alternative 3′ splice site led to dysregulation of gene expression. To clarify the evolutionary history of scorpions based on hyaluronidase mol. phylogenetic studies, we used the phylogenetic tree to deduce the origins of this protein in different species of scorpions, spiders and bees. Hyaluronidase gene performed well for divergences and accurately separated closely related species. Our work provides insights into the diversity of Hase proteins during scorpion evolution and may direct further studies consisting of synthesis, purification and recombinant production of Hase proteins.