Aims: To describe a new molecular technique for the assessment of fungal diversity in the air. Methods and Results: Air samples were collected every week in a henhouse in France during a 15-week period. After air sampling, the collecting membrane was diluted, and the liquid was used for subsequent cultivation and molecular analysis: PCR-temperature temporal gradient electrophoresis (TTGE), which has already been used for the identification of fungal species in air samples and PCR-denaturing high-performance liquid chromatography (D-HPLC), a new technique for the analysis of complex microbial populations. D-HPLC profiles were reproducible from run-to-run, and several fungal organisms could be identified at the species level by sequencing. Conclusions: PCR-D-HPLC enabled the identification of fungal species (both Ascomycota and Basidiomycota) that may be encountered in air. The new technique allowed the detection of more fungal species than did the PCR-TTGE technique. However, some fungal species were detected only by PCR-TTGE, suggesting that PCR-D-HPLC and PCR-TTGE are complementary. Significance and Impact of the Study: PCR-D-HPLC represents a considerable saving in time over currently available procedures for detection and identification of fungal organisms in air. However, the fungal diversity detected by PCR-D-HPLC or by PCR-TTGE was lower than that revealed by culture.