Abstract The purpose of this study was to evaluate the application of previously described Pythium insidiosum- and Lagenidium-specific nested PCR assays to the detection of oomycete DNA in animal tissues. DNA was extracted from 15 frozen and 10 ethanol-fixed tissues obtained from six animals with pythiosis, five animals with lagenidiosis, one animal with nonoomycotic skin disease and two animals without skin disease. First-round PCR, which utilized universal fungal primers ITS1 and ITS2P, amplified a single product of the expected size for each of the P. insidiosum- and Lagenidium-infected tissues, but not for tissues obtained from animals without fungal disease. Second-round PCR using the P. insidiosum-specific primers PI1 and PI2 produced a single 105-bp product for the P. insidiosum-infected tissues, but not for any of the other tissues. Second-round PCR using the Lagenidium-specific primers LAG1 and LAG2 produced a single 76-bp product for the Lagenidium-infected tissues, but not for any of the other tissues.