Cambarus (Puncticambarus) johni is a new species of crayfish from the eastern Blue Ridge foothills and western Piedmont Plateau of North Carolina, where it occurs in streams in the upper Broad, Catawba, and Yadkin-Pee Dee river basins. It shares similarities with other stream-dwelling species of the subgenus, but is distinguished by a suite of characters that includes: an epistome with a weakly curved zygoma that is wider than the space between the renal pores; a dorsal carapace, rostrum and abdomen densely pitted with punctations; a broad, extremely punctate areola; a strong, curved, cephalomesial flange on the ventral surface of the coxa of the fourth pereopod of adult males; and an adult color pattern that includes a dark saddle on the carapace and a pair of dark dorsolateral stripes on the abdomen.