We describe a new species of the genus Leptobrachium from the Khao Laem Mountain, Suan Phung District, Ratchaburi Province, Tenasserim Region, western Thailand, based on molecular and morphological evidences. The new species, Leptobrachium tenasserimense sp. nov., can be distinguished from all other congeners by the following combination of characters: (1) adult SVL of 41.4–58.8 mm in males and 54.7–58.6 mm in females; (2) rounded finger and toe tips; (3) relative finger lengths: II<IV<I<III; relative toe lengths: I<II<V<III<IV; (4) toe webbing thick and well developed; (5) inner metatarsal tubercle small; (6) iris bicolored, black ventrally and turquoise dorsally, with light blue sclera; (7) dorsum brown to grey with distinct darker markings edged with brown; (8) belly and limbs ventrally whitish with contrasting confluent black reticulations; (9) tympanum mostly free of dark marking; (10) narrow dark canthal stripe present; (11) lateral row of dark spots absent; (12) limbs dorsally with distinct dark bars; tibia with four to five dark transverse bars; (13) dense dark reticulation or large dark blotch at groin continuing to ventral and posterior sides of thighs; (14) femoral gland in shape of large white blotch; (15) males with single vocal sac, mature males lack lip spinules. Our study provides further evidence for a hidden biodiversity of montane areas of Tenasserim Region on the border of Thailand and Myanmar.