Mesophotic coral ecosystems (MCEs) extend from 30 to 150 m in depth and support diverse communities of marine organisms. We investigated changes in the structure of mesophotic reef fish assemblages (27–100 m) in the Northwestern Hawaiian Islands (NWHI) along depth and geographical gradients using open- and closed-circuit trimix diving. There were clear changes in the assemblage structure from the southeastern to the northwestern end of the NWHI and from shallow to deep waters. Interactive effects of depth and location were also detected. MCEs in the NWHI can be treated as three regions: southeastern and mid regions primarily separated by the presence and absence, respectively, of the introduced species Lutjanus kasmira, and a northwestern region where fish assemblages are largely composed of endemic species. These spatial patterns may be explained, at least in part, by differences in temperature among the regions.