A new species of Trichomycterus endemic to the Ribeira de Iguape River basin, southeastern Brazil, was studied based on morphological and molecular evidence. This species had an outer layer of coloration composed of scattered, round, black or dark-brown spots smaller or equivalent in size to the circumference of the eye; eight pectoral-fin rays; 28-29 opercular odontodes; 54-56 interopercular odontodes; and supraorbital line of the laterosensory system not interrupted, with pores s2 absent. Two other species of Trichomycterus from the Ribeira de Iguape River basin are recorded, and their taxonomic status is discussed: Trichomycterus alternatus and Trichomycterus jacupiranga were not differentiated using molecular analysis but may be consistently distinguished based on morphology. The phylogenetic relationships of the co-occurring species, T. alternatus and Cambeva zonata, were inferred using mitochondrial data, reinforcing the taxonomic status of these recently revised species that have a complex taxonomy. In addition, a new combination for Trichomycterus taroba with its inclusion in the genus Cambeva is recommended.