As a key component of the largest subunit of the splicing machinery, SF3B1 plays essential roles in eukaryotic growth and development. However, only a few studies have focused on the evolutionary features and functions of this protein in plants. In this study, with the assistance of a bioinformatic analysis, we determined the complete coding sequence of the gene encoding the pine SF3B1 protein using RT-PCR and DNA sequencing. The evolutionary features of SF3B1 proteins were further examined based on a phylogenetic tree of SF3B1 homologous proteins from different eukaryotes, along with comprehensive comparisons of their functional domains, conserved motifs, and cis-regulatory elements and the structures of the corresponding genes. Furthermore, the effects of the splicing modulator GEX1a on several plant species were analysed, confirming that the re-identified SF3B1, with a full-length HEAT repeat domain, is expressed and functions in pre-mRNA splicing regulation in pines. In summary, we conducted a systematic cross-species comparison of SF3B1 homologous proteins, with an emphasis on complete sequence determination and the functional confirmation of pine SF3B1, illustrating the conservation of homologous proteins in plants. This study provides a valuable reference for understanding functional and regulatory mechanisms, as well as the potential applications of SF3B1.