Pimodivir exerts an antiviral effect on the early stages of influenza A virus replication by inhibiting the cap-binding function of polymerase basic protein 2 (PB2). In this study, we used a combination of sequence analysis and phenotypic methods to evaluate pimodivir susceptibility of influenza A viruses collected from humans and other hosts. Screening PB2 sequences for substitutions previously associated with reduced pimodivir susceptibility revealed a very low frequency among seasonal viruses circulating in the U.S. during 2015-2020 (<0.01%; 3/11,934) and among non-seasonal viruses collected in various countries during the same period (0.2%; 18/8971). Pimodivir potently inhibited virus replication in two assays, a single-cycle HINT and a multi-cycle FRA, with IC50 values in a nanomolar range. Median IC50 values determined by HINT were similar for both subtypes of seasonal viruses, A (H1N1)pdm09 and A (H3N2), across three seasons. Human seasonal viruses with PB2 substitutions S324C, S324R, or N510K displayed a 27-317-fold reduced pimodivir susceptibility. In addition, pimodivir was effective at inhibiting replication of a diverse group of animal-origin viruses that have pandemic potential, including avian viruses of A (H5N6) and A (H7N9) subtypes. A rare PB2 substitution H357N was identified in an A (H4N2) subtype poultry virus that displayed >100-fold reduced pimodivir susceptibility. Our findings demonstrate a broad inhibitory activity of pimodivir and expand the existing knowledge of amino acid substitutions that can reduce susceptibility to this investigational antiviral.