Several factors negatively affecting plant growth and production. Abiotic stresses play a vital role against many plant species. Among various abiotic stresses, heavy metals provide one of the most harmful effects on plant performance. Thus, this study was designed to establish an efficient in vitro protocol for chromium (Cr) toxicity tolerance screening in banana. One of the most important commercial banana cultivars (i.e., Grand Nain) was used. Five different concentrations of potassium dichromate (i.e., 50, 100, 200, 400 and 600 ppm) were tested along with free chromium medium as a control. Analysis of variance showed highly significant differences among Cr concentrations, affecting shoot length and number of shoots per explants, compared to control. In addition, results clearly showed the harmful effects of chromium on both shoot length and number of shoots per explant. In this regard, the percentage of reduction in shoot length due to Cr treatment ranged from 5.77 to 84.09% regarding treatment with 50 and 600ppm, respectively. Moreover, the number of shoots per explant was decreased by a range of 6.00 to 86.48% caused by treatment of 50 and 600ppm, respectively. According to these results and the analysis of variance, it is recommended to use the concentration of 400ppm potassium dichromate for in vitro screening of chromium tolerance in banana. The established protocol is efficient and of great importance and could be used for selecting chromium-tolerant genotypes in banana and other species.