As one of the largest families of transcription factors, the R2R3-MYB family plays a significant role in plant growth, development, and response to hormone and environmental stress. To explore its evolutionary mechanism and potential function in Ginkgo biloba, a gymnosperm of great economic and ecological value, we presented a comprehensive analysis of the R2R3-MYB genes in ginkgo. Sixty-nine GbR2R3-MYB genes were identified and these genes could be classified into 33 groups based on the characteristics of the amino acid sequence of the R2R3-MYB domain and gene structure. Syntenic analyses indicated that few tandem and segmental duplications possibly resulted in the contraction of the GbR2R3-MYB gene family. Based on the transcriptome data, expression profiles of eight different tissues and different developmental stages of leaf and kernel showed that GbR2R3-MYB genes had distinct temporal and spatial expression characteristics. Specific expression patterns of the sixteen GbR2R3-MYB genes were also identified in response to different abiotic stresses and hormonal exposures. Further investigation revealed that GbR2R3-MYB19 was located in the nucleus and possessed transcriptional activity, implying its potential roles in the regulation of multiple biological processes. Our findings provide a robust basis for future comprehensive evolutionary and functional analyses of GbR2R3-MYB genes in ginkgo.