Phosphonates, molecules containing direct carbon-phosphorus bonds, compose a structurally diverse class of natural products with interesting and useful biological properties. Although their synthesis in protozoa was discovered more than 50 y ago, the extent and diversity of phosphonate production in nature remains poorly characterized. The rearrangement of phosphoenolpyruvate (PEP) to phosphonopyruvate, catalyzed by the enzyme PEP mutase (PepM), is shared by the vast majority of known phosphonate biosynthetic pathways. Thus, the pepM gene can be used as a molecular marker to examine the occurrence and abundance of phosphonate-producing organisms. Based on the presence of this gene, phosphonate biosynthesis is common in microbes, with ∼5% of sequenced bacterial genomes and 7% of genome equivalents in metagenomic datasets carrying pepM homologs. Similarly, we detected the pepM gene in ∼5% of random actinomycete isolates. The pepM-containing gene neighborhoods from 25 of these isolates were cloned, sequenced, and compared with those found in sequenced genomes. PEP mutase sequence conservation is strongly correlated with conservation of other nearby genes, suggesting that the diversity of phosphonate biosynthetic pathways can be predicted by examining PEP mutase diversity. We used this approach to estimate the range of phosphonate biosynthetic pathways in nature, revealing dozens of discrete groups in pepM amplicons from local soils, whereas hundreds were observed in metagenomic datasets. Collectively, our analyses show that phosphonate biosynthesis is both diverse and relatively common in nature, suggesting that the role of phosphonate molecules in the biosphere may be more important than is often recognized.