Diazotrophic microorganisms introduce biologically available nitrogen (N) to the global N cycle through the activity of the nitrogenase enzyme. The genetically conserved dinitrogenase reductase ( ) gene is phylogenetically distributed across four clusters (I-IV) and is widely used as a marker gene for N fixation, permitting investigators to study the genetic diversity of diazotrophs in nature and target potential participants in N fixation. To date there have been limited, standardized pipelines for analyzing the functional gene, which is in stark contrast to the 16S rRNA gene. Here we present a bioinformatics pipeline for processing amplicon datasets - NifMAP (" MiSeq Illumina Amplicon Analysis Pipeline"), which as a novel aspect uses Hidden-Markov Models to filter out homologous genes to . By using this pipeline, we evaluated the broadly inclusive primer pairs (Ueda19F-R6, IGK3-DVV, and F2-R6) that target the gene. To evaluate any systematic biases, the gene was amplified with the aforementioned primer pairs in a diverse collection of environmental samples (soils, rhizosphere and roots samples, biological soil crusts and estuarine samples), in addition to a mock community consisting of six phylogenetically diverse members. We noted that all primer pairs co-amplified homologs to varying degrees; up to 90% of the amplicons were homologs with IGK3-DVV in some samples (rhizosphere and roots from tall oat-grass). In regards to specificity, we observed some degree of bias across the primer pairs. For example, primer pair F2-R6 discriminated against cyanobacteria (amongst others), yet captured many sequences from subclusters IIIE and IIIL-N. These aforementioned subclusters were largely missing by the primer pair IGK3-DVV, which also tended to discriminate against Alphaproteobacteria, but amplified sequences within clusters IIIC (affiliated with Clostridia) and clusters IVB and IVC. Primer pair Ueda19F-R6 exhibited the least bias and successfully captured diazotrophs in cluster I and subclusters IIIE, IIIL, IIIM, and IIIN, but tended to discriminate against Firmicutes and subcluster IIIC. Taken together, our newly established bioinformatics pipeline, NifMAP, along with our systematic evaluations of primer pairs permit more robust, high-throughput investigations of diazotrophs in diverse environments.