Anthocyanin is an important pigment that affects plant color and nutritional quality. MYBs play an important role in plant anthocyanin synthesis and accumulation. However, the regulatory function of MYB transcription factors in anthocyanin synthesis in flax flowers is still unclear. In this study, 402 MYB transcription factors were identified in the flax genome. These MYB members are unevenly distributed on 15 chromosomes. The R2R3-LuMYB members were divided into 32 phylogenetic subfamilies. qRT-PCR analysis showed that seven R2R3-LuMYB genes in the adjacent subfamily of the evolutionary tree had similar expression patterns, among which LuMYB216 was highly expressed in the petals of different colors. Moreover, gene editing of LuMYB216 in flax showed that the petal color, anther color and seed coat color of mutant plants were significantly lighter than those of wild-type plants, and the anthocyanin content of lumyb216 mutant plants was significantly reduced. Correlation analysis indicated that LuMYB216 was significantly positively correlated with the upstream regulator bHLH30. This study systematically analyzed the MYB gene family in flax, laying a foundation for studying the regulation of LuMYB216 in flax flower anthocyanin synthesis.