A new species of the genus Atelopus, Atelopus fronterizo sp. nov. , from eastern Panama is described herein based on molecular, morphological, and bioacoustic evidence. The new species can be distinguished from its congeners occurring in the region by a combination of the following characters: (1) phalangeal reduction in thumb; (2) SVL (females only) (35.1–50.1; n=13), HW/SVL (0.23–0.34; n=59), EYND/HW (0.27–0.39; n=60), TIBL/SVL (0.41–0.56; n=58), and HAL/SVL (0.22–0.28; n=49); (3) dorsal color pattern with green or yellow background and extensive dark olive blotches forming transversal bands or mottling; (4) advertisement call duration 176–235 ms with 19–34 pulses, average pulse rate 131.69 pulses/s, and dominant frequency 2 422.50–2 606.50 Hz. The new species is nested within the Central American clade of Atelopus. The minimum Kimura‐2‐parameter (K2P) genetic divergence between Atelopus fronterizo sp. nov. and its most phylogenetically similar congeners (A. certus and A. glyphus) is >2.6% for 16S and >4.9% for COI (
). The phylogenetic relationship is strongly supported by ultrafast bootstrap values for the maximum-likelihood trees of both genetic markers (16S, 96; COI, 100,
). Bayesian analysis of the concatenated sequences resulted in a tree with similar topology and high posterior probability support (0.99; Supplementary Figure S1). In addition, haplotype networks inferred from COI and 16S (Supplementary Figure S2) showed a well-separated clade containing the new species (two for COI, four for 16S). The number of mutational steps between haplotypes for the new species samples is very low (1–4 in 16S; one in COI), and the minimum number of mutational steps from the nearest species is nine for 16S (distance to A. certus) and 28 for COI (distance to A. glyphus).