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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 (<0.03%; 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 IC 50 values in a nanomolar range. Median IC 50 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 by HINT. 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. • PB2 sequence analysis and phenotypic assays were used to assess pimodivir susceptibility of influenza A viruses. • Naturally occurring resistance to pimodivir is low in seasonal and non-seasonal influenza A viruses. • Pimodivir potently inhibited replication of widely diverse viruses in a single-cycle HINT or a multi-cycle FRA assays. • Pimodivir was effective against pandemic potential avian A(H5N6) and A(H7N9) viruses. • A rare substitution PB2–H357N conferring >100-fold reduced drug susceptibility was identified in an A(H4N2) poultry virus. [ABSTRACT FROM AUTHOR]