The present study was designed to develop a reliable experimental protocol for detecting and genotyping amplified fragment length polymorphism (AFLP) markers in the commercially important edible mushroom Hypsizygus marmoreus; and to use the markers to evaluate the genetic polymorphisms and phylogenetic relationships among 19 strains of H. marmoreus. Using a set of ten primer pairs, 609 AFLP markers were identified, of which 532 were found segregating among 19 cultivated varieties and laboratory stock strains. An estimate of genetic relationships among the varieties/strains based on the AFLP markers was obtained by primary phylogenetic analysis using UPGMA. Our data demonstrate abundant genetic variation in the natural population of H. marmoreus and facilitate further analysis of genetic divergence within the species. The present study is useful for understanding the genetic structure of this species and uncovering genetic control of economically important traits for efficient breeding programs.