Nitrogen fixation in the sea has attracted the attention of ecologists for decades. Much is known about the habitats in which it occurs and some of the factors that limit N2 fixation activity in different environments, but we still know little about the organisms that fix nitrogen, and what limits the growth and distribution of these organisms in marine environments. Molecular biology technological developments have provided tools for detecting and characterizing N2-fixing organisms in the environment. These techniques hold great promise for unraveling the mysteries and paradoxes of N2 fixation in the sea. In this review, we address the theoretical basis for the use of a molecular approach to N2 fixation, highlight the strengths and weaknesses of the approach, and provide case studies that demonstrate the potential contribution of molecular biology approaches to studies of N2 fixation in the sea.