In contrast to cyanobacteria, the significance of bacteria and archaea in oceanic N2 fixation remains unknown, apart from the knowledge that their nitrogenase (nifH) genes are diverse, present in all oceans and at least occasionally expressed. Non-cyanobacterial nifH sequences often occur as contamination from reagents and other sources, complicating the detection and interpretation of environmental phylotypes. We amplified and sequenced partial nifH gene fragments directly from cell populations sorted by fluorescence activated cell sorting from water collected in the North Pacific Subtropical Gyre (NPSG). Sequences recovered (195 total) included presumed heterotrophic or photoheterotrophic non-cyanobacterial nifH phylotypes previously unreported in the NPSG. A nifH sequence previously found in the South Pacific Gyre (HM210397) was exclusively recovered from sorted picoeukaryote populations, and was detected in water column samples using quantitative PCR (qPCR), with 60% of samples detected in the > 10 μm size fraction in addition to the 0.2–10 μm size fraction. A novel cluster 3-like nifH sequence was also recovered from discrete cell sorts and detected by qPCR in environmental samples. This approach enables the detection of rare nifH phylotypes, identifies possible associations with larger cells or particles and offers a possible solution for distinguishing reagent contaminants from real microbial community components.