The variability within Aspergillus fumigatus Fresenius and related species was examined using macro-, micro-morphology, growth temperature regimes and extrolite patterns. In addition, DNA analyses including partial beta-tubulin, calmodulin and actin gene sequences were used. Detailed examination of strains, considered as A. fumigatus earlier, showed that they could be divided into four groups including A. fumigatus sensu stricto, A. lentulus and two new species. The intraspecific genetic variability within A. fumigatus sensu stricto was low, the sequence differences among 23 strains of the species was at most two bases in each partial beta-tubulin and calmodulin gene. However, intraspecific morphological diversity within the species was high and delineation of the species was equivocal. Therefore, beta-tubulin and calmodulin gene sequences could be critical determinants for the delineation of the A. fumigatus sensu stricto species. A. lentulus including isolates from clinical origin, Korean soil and from a dolphin clustered into an isolated group based on beta-tubulin, calmodulin and actin gene sequences, differing from A. fumigatus by morphological characters, growth temperature and extrolite profile. A. lentulus produces the extrolites auranthine, cyclopiazonic acid, a dimeric indole of unknown structure, neosartorin, some pyripyropens, terrein and some tryptoquivalins and tryptoquivalons. Two pair of isolates (CBS 117194, 117186 and 117520, 117519) clustered into separate groups from A. fumigatus and the other Aspergillus section Fumigati species, including the teleomorph Neosartorya, are proposed as two new species. A. fumigatiaffinis spec. nov. produces the extrolites auranthine, cycloechinulin, helvolic acid, neosartorin, palitantin, pyripyropens, tryptoquivalins and tryptoquivalons, and A. novofumigatus spec. nov. produces the extrolites cycloechinuline, helvolic acid, neosartorin, palitantin and terrein.