To get insight of MYB genes in strawberry, we used bioinformatics methods to screen the MYB genes from the Fragaria vesca genome database, and further analyzed gene structure, conserved motif, chromosomal location, phylogenetic analysis and expression in the period of the development of anther. Our results showed that three types of MYB transcription factors, including 105 R2R3-MYB, 4 MYB3R, 1 MYB4R, were identified. These genes unevenly distributed on 7 chromosomes, e.g., that chromosome Fvb5 contained the largest number of 26 MYB genes. Some of MYBs formed distinct tandem duplicate gene clusters. Although the protein length of strawberry MYB genes differed, all of MYBs contained conserved R2R3 domain, with characteristic amino acids, especially orderly spaced conservative tryptophan residues (W). Phylogenetic and gene structure analysis showed that strawberry MYB genes were divided into 34 distinct groups (A1-A34). The function prediction of strawberry orthologous genes using Arabidopsis thaliana gene annotation showed strawberry MYB genes were most likely involved in flavone synthesis, cell fate, and biotic and abiotic stresses. Temporal or spatial expression trends of MYB genes during strawberry anther development indicated that FvMYB2, FvMYB85, FvMYB74, and FvMYB28 had increased expression during 5 period of the development of strawberry anther, which were mainly expressed in the later period of anther development. These results suggested that those MYB genes may play important role during strawberry anther development. Thus, this paper provided information for functional identification of strawberry MYB genes.