The occurrence of swine H1N1 pandemic was unexpected because our previous focus was concentrated on highly pathogenic avian H5N1 outbreaks. The H1N1 pandemic means that cross-species infection and cross-subtype mutation is not as rare as we had previously thought, and the barriers between species and between subtypes are not strong for influenza A virus. In this study, we use ANOVA to determine if there are barriers between species and between subtypes in the matrix protein 1 family from influenza A virus. The results show that the inter-species/subtype variations are generally much smaller than the intra-species/subtype ones, indicating that the barriers between species and between subtypes are not strong for influenza A viruses, which provides statistical evidence for cross-species infections and cross-subtype mutations.