Juveniles of two Acentrogobius species collected in a mangrove estuary in Sikao Creek, southern Thailand, were identified by morphological and molecular methods. A total of 1315 Acentrogobius specimens were collected and grouped into types A (n = 1107, 4·4–12·0 mm standard length, LS) (melanophore absent or indistinct on posterodorsal contour of caudal peduncle; two rows of melanophore blotches on lateral midline) and B (n = 208, 4·8–12·6 mm LS) (distinct melanophore on posterodorsal contour of caudal peduncle; a single row of melanophore blotches on lateral midline). Based on the reverse series method, the melanophore patterns of larger juveniles were linked with the smallest specimens possessing adult characters. The homogeneities of mitochondrial cytochrome b region sequences between the two juvenile types and adult Acentrogobius species collected in the study area indicated type A to be A. kranjiensis (homogeneity between type A and A. kranjiensis: 99·3–100%), and type B to be A. malayanus (homogeneity between latter 98·1 and 99·7%). No Acentrogobius juveniles were collected from the surf zone outside the creek mouth, both species apparently spending most of their life histories within the estuarine habitat. During their pelagic phase, A. kranjiensis and A. malayanus dispersed in the upper, middle and lower reaches of the creek. On the other hand, occurrence patterns during the benthic phase of A. kranjiensis and A. malayanus differed, the former showing upstream movement and the latter downstream movement with growth. These results emphasize the necessity of analysing early fish life histories at the species level, and the collaboration between morphological and molecular methods should prove valuable in accurately identifying of larvae and juveniles.