Influenza A virus contains eight RNA segments and encodes 10 viral proteins. However, an 11th protein, called PB1-F2, was found in A/Puerto Rico/8/34 (H1N1). This novel protein is translated from an alternative open reading frame (ORF) in the PB1 gene. We analyzed the PB1 gene of 42 recent influenza A isolates in Taiwan, including 24 H1N1 and 18 H3N2 strains. One H1N1 isolate and 17 H3N2 isolates contained the entire PB1-F2 ORF of 90 residues, three amino acids (aa) longer than the PB1-F2 of A/Puerto Rico/8/34 at the C terminal. The one remaining H3N2 isolate encoded a truncated PB1-F2 with 79 residues. The other 23 H1N1 isolates contained a truncated PB1-F2 of 57 aa. Phylogenetic analysis of both the HA and the PB1 genes showed that they shared similar clustering of these Taiwanese isolates, suggesting that no obvious reassortment occurred between the two genomic segments.