Two new miniature species of the trichomycterid genus Tridens are described from the Madeira River drainage, Acre and Rondônia States, Brazil. Until this work, Tridens was a monotypic genus composed solely of Tridens melanops, from the Putumayo/Içá River drainage, upper Amazonas River basin. Tridens vitreus sp.n. is known from upper and middle Madeira River drainage and differs from all other congeners by the lack of pelvic fins and girdle and by vertebra and dorsal-fin ray counts. Tridens chicomendesi sp.n. is known from Abunã River, middle Madeira River drainage and is distinguished from all other congeners by the number of vertebrae, dorsal-fin ray count and anal-fin base colouration pattern. Tr. chicomendesi sp.n. is further distinguished from T. vitreus by a combination of character states regarding the position of urogenital opening, dorsal-fin position, anal-fin position, maxillary barbel length, number of premaxillary teeth, numberof dorsal-fin rays, number of anal-fin rays, number of lateral-line system pores, frontal bone anatomy, degree of ossification of maxilla, anatomy of quadrate-hyomandibular joint, size of posterodorsal process of hyomandibula, length of opercular patch of odontodes, number of interopercular odontodes, proportion of upper hypural plate cartilage relative to its area ; by the absence of a proximal, distal and ventral cartilages on ventral hypohyal; by the absence of a lateral process on basibranchial 4; by the presence of a cartilage block on the lateral process of autopalatine, the presence of a well-developed ossification on proximal margin of ventral hypohyal, the presence of hypobranchial foramen; and by the presence of an anterior cartilaginous joint between quadrate and base of posterodorsal process of hyomandibula. This work represents the first species description for the subfamily Tridentinae in more than 30 years and for the genus Tridens since its original description in 1889.