Cladomorphus petropolisensis sp. nov., a new species of stick insect from Petrópolis, Rio de Janeiro, Brazil, is herein described and compared to the other sympatric species, C. phyllinus Gray, 1835 (Phasmatidae, Cladomorphinae). The description of the new species is supported by morphological and molecular evidence. Kimura-2-parameter (K2P) intraspecific COI divergences among the holotype of C. petropolisensis sp. nov. and C. phyllinus individuals ranged from 2.9% to 4.4%, which are suggestive of distinct species, especially when considering that all Cladomorphus individuals studied were collected in the Petrópolis municipality. The new species can be distinguished from C. phyllinus Gray, 1835 by several characteristics: smaller size, the presence of two spines on the hind femora, the relative longer length of the ovipositor, and spiny tegument, especially in the mesonotum, sculpturing of the operculum of the egg.