The myeloblastosis (MYB) superfamily, which has diverse functions in plants including regulating responses to biotic and abiotic stresses, is one of the largest families of transcript factors (TFs). In this study, we have conducted genome-wide characterization of the MYB family in sunflower (Helianthus annuus L.). A total of 245 genes were identified as candidate MYB genes, which were classified into 10 groups. Genomic localization and paralogs of HaMYB genes mapped the 245 genes on 17 chromosomes with collinearity relationship among genes. Conserved domain, gene structure, and evolutionary relationships of HaMYBs were established and analyzed. Seedlings of sunflower cultivars TK0409 (a sensitive cultivar to Orobanche cumana infection) and JY207 (a resistant cultivar) were imposed to three levels (0, 10%, and 20%) of simulated drought stress treatments using polyethylene glycol (PEG 6000), three levels (0, 150, and 300 mM NaCl) of salinity treatments and two levels (presence and absence) of Orobanche cumana biotic stress treatments. The expression patterns of 55 selected HaMYB genes were investigated for diﬀ;erent stresses. The results from qRT-PCR analyses revealed that many MYB genes in sunflower had significant differences in expression under the different stress treatments, which highlighted the potential of using HaMYB genes as targets for improving stress tolerance in sunflower. Interestingly, under abiotic stress, HaMYB16.1 exhibited higher expression level under simulated drought stress by PEG but was extremely down-regulated under salt stress both in roots and leaves, the transcript levels of the MYB genes HaMYB15.6, HaMYB15.13, HaMYB15.14, and HaMYB58 were significantly enhanced with the increase of salinity concentration in roots, while the expression of HaMYB15.13, HaMYB52.2 and HaMYB52.4 in leaves were also considerably enhanced with the increase of PEG concentration from 10% to 20%. However, under biotic stress, HaMYB3.1 and HaMYB15.12 exhibited higher expression in both roots and leaves of cv. TK0409 but low level of expression in both roots and leaves of cv. JY207. These results indicated the active involvement of MYB genes in response to biotic and abiotic stresses. With this better knowledge on genome-wide identification and expression analyses of MYB gene family in sunflower, this study provides not only important information for stress response mechanism in the crop but also candidate genes for the future improvement of this crop.