Knowledge about the mode of gene action of in vitro traits, which directly contributes towards higher response to anther culture in any crop like summer like summer squash helps to formulate the genetic basis for improvements. Therefore, this investigation aimed to evaluation of a set of diallel crosses from four Squash varieties for their response to anther culture. Subsequently, nature of gene action of these traits could be estimate to determine the proper breeding program for improvement anther culture response. The data were recorded on the following traits: Responding anthers, Callus weight and Shoot ratio. The statistical analysis was made for the obtained data and the results could be summarized in the following: Significant testes on the mean squares of genotypes were highly significant for callus weight / responded anther and shoot ratio, while it was insignificant for responding anther. This result revealed that the comparisons between these genotypes as well as the partition of genetic variance to it's components are valid. No crosses exhibited positive significant heterosis for responding anthers. On the other hand, two out of six crosses exhibited significant positive heterosis for callus weight which were 23.17 % ( White Bush ×  Eskandrani ) and 42.56 % (Eskandrani × White Bush Scallop). While, in the case of shoot ratio, three out of six crosses exhibited positive and significant heteroic values which were 45.87 % ( White Bush × Eskandrani ), 73.02 % (Eskandrani × White Bush Scallop) and 92.73% ( Baladi × White Bush Scallop). In addition, the results revealed White Bush × Eskandrani and Eskandrani × White Bush Scallop were the best combinations for calli weight / responding anthers. Furthermore, the combination between Baladi × White Bush Scallop (P₃ × P₄) was the best regenerable, which had positive and significant SCA effect value for shoot ratio. This finding indicates these combinations  are the best from this set of genotypes for anther culture purpose in Squash. In addition, the results revealed that the magnitude values of dominance genetic variance were positive and larger than the corresponding values of additive genetic variance were positive and larger than the corresponding values of additive genetic variance for the studied in vitro traits. These traits were mainly controlled by dominance genetic variance. This result could be emphasized by dominance degree ratio, which were more than unity, revealing the importance of over dominance in the genetic expression of these traits. In conclusion, from the previous results, which revealed the predominance of non-additive gene action in the gene action in the genetic expression of the studied in vitro traits. These traits were mainly controlled by over dominance genes. It could be recommended the hybrid production as breeding programme for improvement these traits.