The long-term preservation of fungi has been carried out using several different methods, depending on resources and laboratory. Isolates have been deposited in culture collections for preservation and storage for periods of many years. Despite the widespread use of preservation regimes, little attention has been paid to the poststorage stability of the physiological and genetic characters of strains. The loss of viability of a biological control agent or the failure of an isolate to produce a secondary metabolite pivotal in the production of drugs or food could result in substantial economic loss for the manufacturing organisation. In this investigation the effects of five preservation protocols on fungal characters were assessed: continual sub-culture, lyophilisation, storage in water, storage at -20°C and cryopreservation in liquid nitrogen The physiology and genetic stability of three species of economically important fungi (Metarhizium anisopliae, Fusarium oxysporum and Serpula lacrymans) were examined by analysis of culture characteristics, secondary metabolite profiling, extracellular enzyme tests and PCR fingerprinting over a two-year testing period. It was found that preservation regime can influence the resultant characters of the test fungi. Radial growth rate and conidial production was changed from the original isolates after preservation and storage. Secondary metabolite profiles from all of the test fungi were susceptible to change by the preservation protocols assessed. Production of some metabolites was lost, whereas others remained stable after preservation and storage. Extracellular enzyme production was also affected in a similar way. For example, some replicates of an isolate of Metarhizium anisopliae lost ~-galactosidase activity after one year of preservation. Genetic stability was also compromised is some isolates. Polymorphisms were detected after PCR fingerprinting with a micro-satellite primer in replicates of two isolates of Metarhizium anisopliae that had been stored for one and two years by cryopreservation and lyophilisation and in two replicates of an isolate of Fusarium oxysporum maintained by continual sub-culture for sixteen weeks. The results indicate that response to preservation and storage is species- and strain-specific. Therefore, there is a need to develop new and existing preservation regimes with emphasis on strain-specific criteria. Scientists should preserve their important isolates by more than one preservation method to protect organisms from the stresses encountered during preservation and storage.