A real-time quantitative polymerase chain reaction (qPCR) assay targeting two different clades of Trichodesmium spp. was applied to samples from depth profiles in the Atlantic and Pacific Oceans. CladeEEE (T. thiebautii, T. tenue, T. hildebrandtii, and T. pelagicum) was typically more numerous in the open ocean than clade I (T. erythraeum and T. contortum). Concentrations of both clades were higher in the Atlantic than in the Pacific during the timeframe of this study. Neither clade I nor cladeEEE concentrations correlated with surface phosphorus (P) or iron (Fe) concentrations, but clade I was distributed in deeper and colder waters than cladeEEE. Sequences of qPCR products included varieties of Trichodesmium previously found in the field but not in culture. Numerical models parameterizing nitrogen fixation are often based on clade I, while cladeEEE was more numerically dominant in this study. Model results and our understanding of Trichodesmium and global nitrogen fixation may be distorted if there are important ecological differences between clades I andEEE.