Nuclear small subunit ribosomal DNA comparative sequence data analysis is a useful tool for assessing phylogenetic relationships. To date, the small subunit ribosomal DNA genes for only two eumycetozoans Physarum polycephalum and Dictyostelium discoideum have been characterized. Polymerase chain reaction amplifications of other eumycetozoans using universal primers were unsuccessful. Therefore, new primers were designed to regions homologous for both Physarum and Dictyostelium. The utility of these primers was demonstrated via amplification of representatives from the dictyostelids, protostelids, myxomycetes, and acrasids.