The novel influenza A(H1N1) virus that emerged recently in Mexico has spread rapidly to many countries and initiated a human pandemic. It would be interesting to determine whether the virus has existed in, or will spread to, the swine population. However, it is difficult to differentiate the virus from some swine influenza viruses. In this study, a SYBR Green I real-time RT-PCR assay was designed for detection and differentiation of influenza A(H1N1) virus from some swine influenza viruses, by comparing the amplification of two pairs of primers corresponding to influenza A(H1N1) virus and some swine influenza viruses, respectively. The assay was evaluated using online analysis, identified influenza viruses and clinical samples. The results indicated that the assay has high sensitivity and specificity to detect influenza A(H1N1) virus, and is able to differentiate it from some swine influenza viruses. This, in turn, could provide essential epidemiological information for risk analysis and decision making in combating the disease, and stimulate research to differentiate pathogens similar to each other using the same method.