Biological nitrogen fixation (BNF) supplies nutrient-depleted oceanic surface waters with new biologically available fixed nitrogen. Diazotrophs are the only organisms that can fix dinitrogen, but the factors controlling their distribution patterns in the ocean are not well understood. In this study, the relative abundances of eight diazotrophic phylotypes in the subtropical North Atlantic Ocean were determined by quantitative PCR (qPCR) of the nifH gene using TaqMan probes. A total of 152 samples were collected at 27 stations during two GEOTRACES cruises; Lisbon, Portugal to Mindelo, Cape Verde Islands (USGT10) and Woods Hole, MA, USA via the Bermuda Time Series (BATS) to Praia, Cape Verde Islands (USGT11). Seven of the eight diazotrophic phylotypes tested were detected. These included free-living and symbiotic cyanobacteria (unicellular groups (UCYN) A, B and C, Trichodesmium, the diatom-associated cyanobacteria Rhizoselinia-Richelia and Hemiaulus-Richelia) and a γ-proteobacterium (Gamma A, AY896371). The nifH gene abundances were analyzed in the context of a large set of hydrographic parameters, macronutrient and trace metal concentrations measured in parallel with DNA samples using the PRIMER-E software. The environmental variables that most influenced the abundances and distribution of the diazotrophic phylotypes were determined. We observed a geographic segregation of diazotrophic phylotypes between east and west, with UCYN A, UCYN B and UCYN C and the Rhizosolenia-Richelia symbiont associated with the eastern North Atlantic (east of 40°W), and Trichodesmium and Gamma A detected across the basin. Hemiaulus-Richelia symbionts were primarily found in temperate waters near the North American coast. The highest diazotrophic phylotype abundance and diversity were associated with temperatures greater than 22. °C in the surface mixed layer, a high supply of iron from North African aeolian mineral dust deposition and from remineralized nutrients upwelled at the edge of the oxygen minimum zone off the northwestern coast of Africa