The button mushroom Agaricus bisporus is cultivated almost worldwide. Its cultivation is standardized and a temperature of 16-19°C is needed during the fruiting period. The development of A. bisporus cultivars able to fruit at higher temperature (FHT) represents a promising alternative to reduce energy costs during cultivation in hot countries as well as in temperate countries during the hot season. A. bisporus var. burnettii is able to fruit at 25°C. Understanding the biological mechanisms that underlie such a thermo-tolerance is a prerequisite to further development of breeding strains. The foundation of the FHT ability of the var. burnettii was dissected using a combination of candidate gene approaches and genetic tools. Transcript profiling of A. bisporus var. burnettii at two developmental stages (primordium P and sporophore SP) under two fruit-producing temperature conditions (17°C and 25°C) were established by cDNA-AFLP. The expression patterns were more similar within the same stage at the two different temperatures rather than between stages under the same temperature. Only one transcript-derived fragment (TDF) sequence differentially expressed between temperatures was recovered but it could not be further characterized. Twenty-nine TDF sequences differentially expressed between development stages were obtained. The phenotypic assessment of an intervarietal A. bisporus var. bisporus×A. bisporus var. burnettii progeny demonstrated the complex inheritance of the FHT trait. Two quantitative trait loci (QTL) involved in the number of fruit bodies yielded at 25°C were found on LG II and LG VI. Two functional candidate genes known to be potentially involved in A. bisporus thermo-tolerance, a heat shock protein (HSP70) gene and a gene coding for a para-aminobenzoic acid synthase (PABA), were found in the vicinity of the QTL on LG II. Several positional candidate genes have been also identified in the confidence interval of the QTL on LG VI and are promising for further fine mapping purpose.