Musty odor owing to 2-methylisoborneol (2-MIB) is a worldwide concern, causing deterioration in the quality of tap water and fish catch. In lakes, a large number of 2-MIB-producing cyanobacteria have been identified to be Pseudanabaena species, but the mechanisms underlying their occurrence have not been revealed. In this study, we investigated the occurrence-related characteristics and taxonomical composition of 2-MIB-producing Pseudanabaena in Lake Ogawara, Aomori Prefecture, Japan, using molecular biology techniques in combination with microscopic observations. In molecular biological analyses, amplicon sequencing and qPCR specific for 2-MIB-producing species were performed. Musty odor-producing Pseudanabaena-like cyanobacteria (MOPPL-cyanobacteria) observed by microscopy mainly occurred during autumn–winter and, to a lesser extent, during spring–summer. There was a positive correlation between the densities of MOPPL-cyanobacteria and 2-MIB-producing strains, but the latter accounted for only 8% of the former, suggesting that a large number of non-MIB-producing strains are included in the microscopy results. At least two 2-MIB-producing Pseudanabaena strains probably exist in Lake Ogawara, and the composition of MOPPL-cyanobacteria is likely to vary at the species/strain level, which cannot be microscopically detected.