Pontodrilus litoralis is a cosmopolitan littoral earthworm known to exhibit bioluminescence. Recently, a congeneric species Pontodrilus longissimus from Thailand was described. These species are sympatric but their burrowing depths on Thai beaches are different. In this study, we examined the in vivo and in vitro bioluminescence properties of P. longissimus and P. litoralis. Mechanical stimulation induced in vivo luminescence in P. litoralis, as reported previously, but not in P. longissimus. In vitro cross-reaction tests between these species revealed the absence of luciferin and luciferase activities in P. longissimus. P. litoralis had strong fluorescence in a coelomic fluid that matches to the spectral maximum of its bioluminescence, but P. longissimus did not. These results suggest that P. longissimus does not have luminescence ability due to the lack of all bioluminescent components, luciferin, luciferase, and light emitter, despite its close relationship to the luminous P. litoralis. The presence of both luminous and non-luminous species in a single genus is uncommon, and our present findings will shed insight on the possible functions of bioluminescence in the earthworm, such as avoiding predation by littoral earwigs.