Barley is a widely adaptable cereal grain, but with a low response from anther cultures to callus induction. In this study, the response to anther culture and the genetic diversity of 16 Iranian and 26 European barley cultivars were evaluated using 20 ISSR (Inter Simple Sequence Repeats) primers. The callus induction phase was investigated based on a completely randomized design with an unequal number of replications. The regeneration phase was studied using a factorial experiment based on a completely randomized design with 3 replications. The factors included the tested cultivars and several growth regulators. All cultivars that responded to anther cultures (8 of 42 cultivars) had European origin. A correlation analysis showed the highest correlation between the callus size and its growth rate. A cluster analysis based on anther culture traits grouped 8 cultivars into 2 classes. The effect of growth regulators was not significant for total regeneration ratio, green and albino plants ratio. The results showed that the efficiency of the anther culture method is largely dependent on the plant genotype. The percentage of polymorphism using ISSR markers ranged from 50 to 78.94. The range of genetic similarity coefficients among barley cultivars was from 0.138 to 0.867. According to the Mantel test, there was no significant correlation between the assumed variation patterns using ISSR molecular markers and anther culture data. The cluster analysis based on the ISSR markers divided 42 barley cultivars into 6 classes, although there was no clear pattern of clustering for differentiation between Iranian and European cultivars. Iranian cultivars showed a higher molecular variation than European ones.