One hundred and forty three airborne Fusarium isolates in chicken houses belonging to seven Fusarium species were analyzed by PCR with Tri5 gene as a specific marker of mycotoxin product . The result of Tri5 gene sequence analysis indicates that the PCR amplification products were 89%-96% identical to the previously reported Tri5 genes, which were all amplified from four F. poae isolates. T-2 toxin and DON was measured by immunoaffinity column and high performance liquid chromatography in Tri5-positive F. poae isolates after being cultured at constant and alternating temperatures. The production of T-2 toxin under alternating temperatures was 14 and 53 times higher than those at constant temperature of 8 °C and 25 °C. No DON was detected under either testing temperature condition. It is concluded that T-2 toxin-producing F. poae isolates were present in poultry houses, and the concentration of T-2 toxin produced by Tri5-positive F. poae isolates was increased under alternating temperatures. The application of Tri5-PCR associated with IMC-HPLC is an effective and accurate method for rapid detection of T-2 and DON mycotoxins.