Abstract: In order to establish an efficient anther culture system of naked barley and explore the synergistic effects of medium optimization and genotypes, this study conducted anther culture using two medium combinations（M₁ and M₂） with five barley materials. And using the optimized medium combination M₂, 49 naked barley materials were cultured in vitro to explore the synergistic effect of medium optimization and genotype screening on improving the efficiency of naked barley anther culture. The results showed that:（1） The combination M₂ effectively enhanced the anther culture efficiency of naked barley. The callus induction rate, green seedling differentiation rate and average anther culture efficiency of M₂ were increased by 9.73, 14.28 and 2.76 percentage points comparing with M₁, respectively.（2） In the combination M₂, the callus was induced from 34 samples. Among them, the hybrid materials performed significantly better than homozygous materials. Line 22-73-1-1 showed the best performance among all the materials, with a callus induction rate of 46.91%, green seedling differentiation rate of 72.37%, and anther culture efficiency of 33.95%.（3） The anther culture efficiency of naked barley varies greatly among different genotypes, showing genotype-dependent characteristics. Flow cytometry analysis showed that diploids accounted for 80% and haploids for 20% in the regenerated plants, without multiploids plants.