In this study, we developed and validated a culture-independent method for diversity surveys to specifically detect endospore-forming Firmicutes. The global transcription regulator of sporulation (spo0A) was identified as a gene marker for endospore-forming Firmicutes. To enable phylogenetic classification, we designed a set of primers amplifying a 602 bp fragment of spo0A that we evaluated in pure cultures and environmental samples. The amplification was positive for 35 strains from 11 genera, yet negative for strains from Alicyclobacillus and Sulfobacillus. We also evaluated various DNA extraction methods because endospores often result in reduced yields. Our results demonstrate that procedures utilizing increased physical force improve DNA extraction. An optimized DNA extraction method on biomass pre-extracted from the environmental sample source (indirect DNA extraction) followed by amplification with the aforementioned primers for spo0A was then tested in sediments from two different sources. Specifically, we validated our culture-independent diversity survey methodology on a set of 8338 environmental spo0A sequences obtained from the sediments of Lakes Geneva (Switzerland) and Baikal (Russia). The phylogenetic affiliation of the environmental sequences revealed a substantial number of new clades within endospore-formers. This novel culture-independent approach provides a significant experimental improvement that enables exploration of the diversity of endospore-forming Firmicutes.