Populations of nitrogen-fixing cyanobacteria in the genus Trichodesmium are critical to ocean ecosystems, yet predicting patterns of Trichodesmium distribution and their role in ocean biogeochemistry is an ongoing challenge. This may, in part, be due to differences in the physiological ecology of Trichodesmium species, which are not typically considered independently in field studies. In this study, the abundance of the two dominant Trichodesmium clades (Clade I and Clade III) was investigated during a survey at Station ALOHA in the North Pacific Subtropical Gyre (NPSG) using a clade-specific qPCR approach. While Clade I dominated the Trichodesmium community, Clade III abundance was >50% in some NPSG samples, in contrast to the western North Atlantic where Clade III abundance was always <10%. Clade I populations were distributed down to depths >80 m, while Clade III populations were only observed in the mixed layer and found to be significantly correlated with depth and temperature. These data suggest active niche partitioning of Trichodesmium species from different clades, as has been observed in other cyanobacteria. Tracking the distribution and physiology of Trichodesmium spp. would contribute to better predictions of the physiological ecology of this biogeochemically important genus in the present and future ocean.