To assess the feasibility of marker-assisted selection in mushrooms, a comparative mapping study between two connected populations of the white button mushroom Agaricus bisporus was performed. The first mapping population had been used already for the construction of the A. bisporus reference linkage map. In the present study, a new linkage map based on the segregation analysis of a second generation hybrid progeny was developed. In order to increase the number of available anchor markers, we developed a conversion procedure of an amplified fragment length polymorphism (AFLP) fragment into sequence-specific PCR marker. Seventeen AFLP-converted markers (ACM) were then used for mapping purpose, among which 14 were common to the two maps. The linkage map presented herein consists of 183 markers (53 cleaved amplified polymorphic sequence, 16 SSR, 17 ACM, 96 AFLP and PPC1 locus), distributed among 13 linkage groups (LG), and covering 851 cM. Thanks to 84 common markers, we have stated that marker order was well conserved, except for LG I; significant unequal recombination rates occurred over the whole genome; regions with markers showing skewed segregation patterns differed between the two maps. Our results suggested a strong impact of the genetic background on recombination ability. Consequences for mushroom breeding are discussed. These maps will facilitate further comparative mapping studies of quantitative trait locus detection.