The herbicide paraquat is used to kill plant tissues and accelerate the growth of quiescent fungal colonists. In this study, freezing was investigated as an alternative to paraquat for the detection of latent infections of Colletotrichum spp. on strawberry petioles. Apparently healthy petioles from field-grown plants and inoculated petioles from greenhouse-grown plants were killed by freezing or exposure to 0.3% paraquat, and incubated in petri dish moist chambers. Previously frozen petioles were treated with 0.525 or 0.0525% sodium hypochlorite and Tween 20 at 18 µl/liter for selected intervals to test the effects of surface disinfestation on detection frequency. After 5 to 7 days of incubation, Colletotrichum acervuli developed much more frequently on freeze- and paraquat-treated petioles than on washed petioles which were not killed. Detection frequencies were similar for paraquat and some freeze treatments, but the latter were negatively affected by prolonged surface disinfestation. Using the petiole freeze method, Colletotrichum acutatum, C. gloeosporioides, and Glomerella cingulata (the teleomorph of C. gloeosporioides) were detected on symptomless petioles of field-grown plants. In addition, C. acutatum and C. dermatium were detected on apparently healthy transplants from northern nurseries. All of these fungi are reported pathogens of strawberry, but not all C. gloeosporioides isolates from frozen petioles were pathogenic in greenhouse bioassays. Freezing is a viable, nonhazardous alternative to paraquat for the detection of latent Colletotrichum infections on strawberry.