A new genus, , and two new species are described based on phylogenetic analyses and phenotypic and ecological characters. The species delimitation was based on concordance of gene genealogies. The cultures were isolated from the roots of mountain laurel () and pitch pine () from the acidic and oligotrophic New Jersey pygmy pine plains; however, they likely have a broader distribution because their internal transcribed spacer (ITS) sequences have high similarity to a number of environmental sequences from multiple independent studies. Based on the phylogeny and phenotypic characters, a new family, Pygmaeomycetaceae, is proposed to accommodate this new lineage in Mucoromycotina. Pygmaeomycetaceae corresponds to Clade GS23, which was identified based on a sequence-only soil fungal survey and was believed to be a distinct new class. Compared with the culture-based methods, we observed that sequence-only analyses tend to overestimate the taxonomic level. Results from this work will facilitate ecological and evolutionary studies on root-associated fungi.