Various incubation conditions (35°C-38°C, 2-7 days) have been used in surveillance studies of the prevalence of avian influenza viruses in wild birds. Here, we studied viral polymerase activity and virus growth kinetics of low-pathogenic avian influenza viruses (LPAIVs) isolated from field samples [A/duck/Hong Kong/365/1978 (H4N6) and A/duck/Nanchang/2-0480/2000 (H9N2)] during incubation at different temperatures (35°C, 37°C, and 39°C) in the allantoic cavity of 10-day-old embryonated chicken eggs (ECE). The higher incubation temperatures (37°C and 39°C) resulted in a significantly higher rate of virus growth, which is most likely a result of increased viral polymerase activity (20%-60%), than was observed at 35°C, and as much as a 100% greater virus yield (as measured by hemagglutination assay) was observed two days after inoculation. Our findings revealed that the optimal activity of the viral polymerase complex, resulting in the highest yield of LPAIV field isolates, could be obtained by incubation for two days in ECE at 37°C and 39°C.