A large scale of EST sequences of Polyporales was screened in this investigation in order to identify EST-SSR markers for various applications. The distribution of EST sequences and SSRs in five families of Polyporales was analyzed, respectively. Mononucleotide was the most abundant type, followed by trinucleotide. Among five families, Ganodermataceae occupied the most SSR markers, followed by Coriolaceae. Functional prediction of SSR marker-containing EST sequences in Ganoderma lucidum obtained three main groups, namely, cellular component, biological process, and molecular function. Thirty EST-SSR primers were designed to evaluate the genetic diversity of 13 natural Polyporus umbellatus accessions. Twenty one EST-SSRs were polymorphic with average PIC value of 0.33 and transferability rate of 71%. These 13 P. umbellatus accessions showed relatively high genetic diversity. The expected heterozygosity, Nei's gene diversity, and Shannon information index were 0.41, 0.39, and 0.57, respectively. Both UPGMA dendrogram and principal coordinate analysis (PCA) showed the same cluster result that divided the 13 accessions into three or four groups.