Abiotic stresses have a significant negative impact on plant growth and production. Among which, toxicity of heavy metals is considered one of the most factors affecting plant growth and agriculture on new reclaimed lands. In this study, the harmful effect of chromium was evaluated on in vitro banana plants of Grand-Nain and Williams-Zeef cultivars based on morphological, physiological, and molecular assessments. Results showed that chromium significantly decreased all studied traits, including plant fresh weight, plant length, number of shoots per explant, and photosynthesis related pigments (chlorophyll and carotenoid content). The percentage of reduction due to chromium treatment ranged from 36.48 to 79.69% in shoot length of Grand Nain and chlorophyll-b in Williams-Zeef, respectively. Both banana cultivars were negatively affected by chromium treatment. However, Williams-Zeef showed higher reduction than that of Grand-Nain in all studied traits. On the other hand, molecular analysis was performed to detect any variation between chromium-treated and untreated plants using inter simple sequence repeats (ISSR) and start codon targeted (SCoT) polymorphism. Results of molecular analysis confirmed the morphophysiological findings, by detection some polymorphic bands due to chromium treatment. In this regard, a total of six polymorphic bands were detected in the two banana cultivars, discriminating treated and control plants. In agreement with morphophysiological results, Williams-Zeef showed more polymorphism (five bands) due to chromium treatment than Grand-Nain (one band). The screening protocol used in this study was efficient and helpful and could be used in successive studies to evaluate other toxicants and with other plant species as well.