Anguillid eels of the genus Anguilla, which have a unique catadromous life history, are widely distributed across many parts of the world. However, little research has been conducted on the behavioural mechanisms of habitat segregation between sympatric species in tropical anguillid eels. To understand the ecological and behavioural mechanisms involved in the life history and migration of tropical anguillid eels, strontium (Sr):calcium (Ca) ratios were examined in otoliths of A. bengalensis bengalensis (41 specimens) and A. bicolor bicolor (130 specimens) collected from ten rivers in northwestern Peninsular Malaysia. The otolith Sr:Ca ratios revealed different habitat use between the two species. The broad range of otolith Sr:Ca ratios and habitat shift found in A. bicolor bicolor suggested that its habitat utilization was opportunistic in environments of varying salinity. A. bicolor bicolor prefers to live in the midstream to downstream areas with tidal influences. A. bengalensis bengalensis, however, was found to only reside in freshwater environments throughout their continental growth. A. bengalensis bengalensis tends to live in upstream area with no tidal influence. Their habitat use, migratory history, and habitat distribution indicate that habitat segregation occurs between the two species, leading to the different habitat preferences in tropical river systems.