Aims: The genetic relationships and conidial tolerances to high and low temperatures were determined for isolates of several Metarhizium species and varieties. Methods and Results: Molecular-based techniques [AFLP and rDNA (ITS1, ITS2 and 5Æ8S) gene sequencing] were used to characterize morphologically identified Metarhizium spp. isolates from a wide range of sources. Conidial sus- pensions of isolates were exposed to wet heat (45 ± 0Æ2°C) and plated on potato dextrose agar plus yeast extract (PDAY) medium. After 8-h exposure, the iso- lates divided clearly into two groups: (i) all isolates of Metarhizium anisopliae var. anisopliae (Ma-an) and Metarhizium from the flavoviride complex (Mf) had virtually zero conidial relative germination (RG), (ii) Metarhizium anisopliae var. acridum (Ma-ac) isolates demonstrated high heat tolerance (c. 70–100% RG). Conidial suspensions also were plated on PDAY and incubated at 5°C for 15 days, during which time RGs for Ma-an and Ma-ac isolates were virtually zero, whereas the two Mf were highly cold active (100% RG). Conclusions: Heat and cold exposures can be used as rapid tools to tentatively identify some important Metarhizium species and varieties. Significance and Impact of the Study: Identification of Metarhizium spp. cur- rently relies primarily on DNA-based methods; we suggest a simple tempera- ture-based screen to quickly obtain tentative identification of isolates as to species or species complexes.