A new species of the suckermouth armored catfish genus Peckoltia is described from the lower Ventuari River, a tributary of the upper Orinoco River in Amazonas State, Venezuela. Specimens of this species were formerly included in the wide-ranging Amazonian species P. vittata, but a recent molecular phylogeny found Orinoco individuals to be distantly related to Amazon Basin individuals spanning the range of P. vittata syntypes. Detailed morphological examination confirmed distinctiveness of Orinoco specimens, and found them to be diagnosable from true P. vittata by having generally greater than 25 teeth (vs. less), spots on the nape (vs. nape lacking spots), the upper lip with two to three black bar-shaped markings in a line like a moustache (vs. lips generally with a hyaline wash), and by the snout having a medial black line disconnected from the moustache markings (vs. medial snout stripe connected to a bar just above the lip). Peckoltia wernekei displays remarkable genetic similarity to its sister species, P. lujani, but differs morphologically by having dentary tooth rows meet at an angle less than 90° (vs. >90°), by having large faint blotches on the abdomen (vs. abdomen with no blotches), by a smaller internares width (21.2–26.6% vs. 28.5–46.5% of interorbital width), and a larger dorsal spine (148.1–178.6% vs. 80.1–134.5% of abdominal length).