The importance of global influenza surveillance using simple and rapid diagnostics has been frequently highlighted. For influenza type B, the need exists for discrimination between the two currently circulating major lineages, represented by virus strains B/Victoria/2/87 and B/Yamagata/16/88, as only one of these lineages is represented in seasonal influenza vaccines. Here, the development and characterization of a low-density DNA microarray (designated BChip) designed to detect and identify the two influenza B lineages is presented. The assay involved multiplex nucleic acid amplification and microarray hybridization of viral RNA. Detection and lineage identification was achieved in less than 8 h. In a study of 62 influenza B virus samples from 19 countries, dating from 1945 to 2005, as well as 5 negative control samples, the assay exhibited 97% sensitivity and 100% specificity. Furthermore, application of a trained artificial neural network to the pattern of relative fluorescence signals resulted in correct lineage assignment for 94% of 50 applicable influenza B viruses, with no false assignments.