Introduction
Influenza B virus (IBV) causes seasonal epidemics and undergoes continuous genetic evolution that may affect vaccine effectiveness. However, data on the molecular epidemiology and long-term evolutionary dynamics of IBV in Saudi Arabia remain limited. Therefore, this study aimed to characterize the genetic diversity, evolutionary dynamics, lineage replacement, and vaccine-related genetic changes of circulating IBVs in Riyadh, Saudi Arabia, during 2010–2025.

Methods
From 2014 to 2020, 311 respiratory specimens were collected from influenza-like illness patients in Riyadh. IBV was detected by reverse transcription polymerase chain reaction (RT-PCR), and complete HA and NA genes were sequenced. Mutations at both nucleotide and amino acid levels were recorded. Glycosylation profiles were analyzed. Phylogenetic comparisons included WHO vaccine reference strains, six IBV isolates generated from specimens collected during 2014–2020, and 31 previously published Riyadh sequences from 2010–2025 to assess the long-term lineage dynamics of IBV in Riyadh.

Results
Of 311 specimens, 88 (28.3%) were influenza A positive and 6 (1.9%) IBV positive. Phylogenetic analysis revealed B/Yamagata lineage viruses in 2015 and B/Victoria lineage viruses in 2020. HA and NA proteins were highly conserved relative to vaccine strains, despite substitutions in antigenic regions (120-loop, 150-loop, 160-loop, 190-helix). A subset of recent B/Victoria isolates (2023–2025) carried the characteristic 164–165 deletion, while B/Yamagata showed lineage-specific glycosylation changes. N-linked glycosylation sites were largely conserved, but gain/loss at HA position 197 occurred in some isolates, potentially affecting antigenicity. Analysis of additional previously published Riyadh sequences from 2010 to 2025 (n?=?31) showed sustained B/Victoria predominance (subclade V1A.3a.2 and V1A.3); no B/Yamagata-lineage viruses were detected among the sequenced isolates. Recent strains remained genetically close to vaccine strains B/Austria/1,359,417/2021 and B/Tokyo/EIS13-175/2025, with only limited amino acid substitutions.

Conclusions
Long-term surveillance (2010–2025) in Riyadh revealed replacement of B/Yamagata by B/Victoria lineage viruses within the available surveillance dataset. Recent isolates (2021–2025) all belonged to the globally dominant V1A.3a.2 subclade. IBVs showed ongoing evolution but remained closely related to WHO-recommended vaccine strains. The predominance of B/Victoria and the non-detection of B/Yamagata within the available dataset underscore the need for continuous molecular surveillance to monitor viral evolution, assess vaccine strain compatibility, and support influenza prevention in Saudi Arabia.