The wheat stripe rust fungus, Puccinia striiformis f. sp. tritici (Pst), does not have a known alternate host for sexual reproduction, which makes it impossible to study gene linkages through classic genetic and molecular mapping approaches. In this study, we compared 4,219 Pst expression sequence tags (ESTs) to the genomic sequence of P. graminis f. sp. tritici (Pgt), the wheat stem rust fungus, using BLAST searches. The percentages of homologous genes varied greatly among different Pst libraries with 54.51%, 51.21%, and 13.61% for the urediniospore, germinated urediniospore, and haustorial libraries, respectively, with an average of 33.92%. The 1,432 Pst genes with significant homology with Pgt sequences were grouped into physical groups corresponding to 237 Pgt supercontigs. The physical relationship was demonstrated by 12 pairs (57%), out of 21 selected Pst gene pairs, through PCR screening of a Pst BAC library. The results indicate that the Pgt genome sequence is useful in constructing Pst physical maps.