This research analyzed the biometrics and reproductive characteristics of a group of Hypancistrus sp. L-333 (King Tiger Plecos) in order to gather biological information and aid the rearing of the species in captivity, thus supporting its natural conservation. This species has a natural distribution that is restricted to the lower Xingu River, and is currently threatened by the hydroelectric projects in the region. The acquired specimens were measured, weighed and classified according to sex based on morphometric characteristics (evidence of secondary sexual characteristics) that were later confirmed by dissection. A total of 32 individuals, 16 males and 16 females were identified. Although the length-weight ratio did not present significant differences, the results revealed that the species has secondary sexual dimorphism. The odontodes were more developed in the mid-lateral region of the body, post-dorsal region, caudal peduncle and in the first ray (ossified) of the pectoral fin in males. Histological analyses of the gonads confirmed that all of the fish were adult individuals. The diameter of the oocytes ranged from 0.14 to 2.0 mm, presented three distinct modes, and were synchronous in more than two groups, which evidences batch spawning. Fecundity was low, between 35 and 54 oocytes in the largest batch. This information is novel and important for the identification of reproductive groups, and is aimed at breeding in captivity so that these resources can be exploited in a sustainable way, without harming the already threatened natural stocks.