Heterometrus yaleensis sp. n. from Sri Lanka, Southern Province, Yale National Park is described and compared with other species of the genus. The presence of a unique dorsointernal carina on the pedipalp chela distinguishes H. yaleensis sp. n. from all other Heterometrus species. Additional information is provided on the taxonomy and distribution of the genus Heterometrus in Sri Lanka, fully complemented with color photos of specimens of both sexes of the new species, as well as of their habitat. In addition to external morphology and hemispermatophore, we also describe the karyotype of H. yaleensis sp. n. (2n=99).