We present a molecular phylogenetic analysis including a survey for overlooked phenotypic characters. Based on both analysis and characters a new cave-dwelling species is described: Tityus (Tityus) spelaeus sp. nov. from the Russão II cave, Posse, state of Goiás, Central Brazil. Characters such as the glandular regions of the female pectinal basal piece and basal middle lamellae of pectines, and the distribution of the ventral setae of telotarsi I–IV proved to be useful to constructing the taxonomy of species and species groups of Tityus. The new species is a member of the Tityus trivittatus species-group of Tityus (Tityus) and can be readily recognized by the immaculate coloration pattern and the more developed glandular region on the female pectinal basal piece. In addition, we provide a discussion of the phylogenetic relationships observed within Tityus, on the relevance of the new phenotypic characters to the modern taxonomy of the genus Tityus, and to the records of Brazilian cave scorpions.