Using atrazine-impregnated Bio-Sep beads as an in situ enrichment method for sampling active soil microbiota, we were able to cultivate taxonomically diverse groups of atrazine degrading bacteria. Most notable among the bacteria isolated were three novel Beta and one novel Alpha-Proteobacteria. Also, a novel lineage within the CFB division was obtained, constituting the first report of the atrazine-degading phenotype within this division. Although not taxonomically novel, previously unreported atrazine-degrading taxa from Alpha-Proteobacteria (Caulobacter), Beta-Proteobacteria (Variovorax, Duganella, Schlegella, and Acidovorax), and Gamma-Proteobacteria (Rahnella and Pantoea) were also isolated. Evidence for metabolic diversity in atrazine catabolism was observed in the collection. Most significantly, the atrazine-chlorohydrolase gene encoded by trz N, was the only known catabolic gene detected in our collection with the exception of the Arthrobacter strains which typically also possessed atzB and atzC, that code for enzymes needed for sequential dealkylation of 2-hydroxy atrazine. No other known genes for the intermediate metabolism were detected in many of the isolates suggesting the presence of alternative degradative pathways for atrazine among soil bacteria. Previously, trz N has only been reported in high GC Gram-positive bacteria but our results revealed that this catabolic gene is much more broadly distributed among classes including the Alpha and Beta-sub classes of the Proteobacteria. The results demonstrate that BioSep beads are a suitable matrix for recruiting a highly diverse subset of the bacterial community involved in atrazine degradation.