Baloxavir marboxil (BXM) is a potent inhibitor of the polymerase acidic (PA) protein of influenza viruses. However, clinical trials predominantly involving influenza A(H1N1) and A(H3N2) infections showed that BXM exhibited a low barrier of resistance. Contrasting with influenza A viruses, BXM-resistant influenza B variants remain poorly documented. We evaluated the impact of I38T/M and E23K PA substitutions, previously reported in influenza A viruses, on in vitro properties and virulence of contemporary influenza B recombinant viruses. Influenza B/Phuket/3073/2013 recombinant wild-type (WT) virus and the I38T, I38M and E23K PA mutants were assessed for their susceptibility to baloxavir acid (BXA), the active metabolite of BXM, by plaque reduction assays in ST6GalI-cells. Luciferase-based minigenome tests were performed to determine polymerase activity. Replication kinetics and genetic stability were evaluated in ST6GalI-cells. Virulence was evaluated in BALB/c mice. The I38T, I38M and E23K substitutions increased BXA IC50s values by 12.6-, 5.5-, and 2.6-fold, respectively, compared to the WT. Minigenome assays revealed a 46% loss of polymerase activity for the E23K substitution vs the WT while the I38T and I38M PA variants retained ≈ 80% of activity. Peak viral titers were comparable for the WT, I38T and I38M recombinants (7.95±0.5, 7.45±0.25 and 8.11±0.28 logTCID50/mL), respectively, whereas it was significantly lower for the E23K mutant (6.28±0.28 logTCID50/mL;P<0.05 vs the WT). In mice, the WT, I38T and I38M recombinants induced mortality rates of 60%, 40% and 100%, respectively and similar lung viral titers were obtained for the three groups at days 3 and 6 p.i.. In conclusion, the fitness of BXA-resistant I38T and I38M PA mutants appears unaltered in contemporary influenza B viruses warranting surveillance for their emergence.