In vitro-stored plant germplasm is usually evaluated by visual analysis of the plant based on subjective characters. To reduce the variability in these evaluations, we developed a digital-image evaluation system for in vitro-stored plantlets. This study compares the standard visual evaluation system with a digital analysis system to determine if digital analysis can effectively quantify the health of diverse Humulus germplasm. Eight cultivars of Humulus lupulus L. were stored on standard Murashige and Skoog (MS) medium with iron alone (EDTA chelated) and on MS iron with 100 or 200 mg l⁻¹ sequestrene 138 iron (EDDHA chelated). Digital images of the upper two nodes of each plantlet were evaluated for red, green, blue, green/red ratio, and modified normalized difference vegetation index (MNDVI=R−G/R G). Evaluation of each plantlet for MNDVI values showed consistent significant differences for all treatments only at the upper node. Significant differences for visual and the MNDVI values among the three iron treatments were observed at the upper node of most of the eight hop cultivars. Regression analysis of the upper node MNDVI values vs. whole-plant visual ratings showed positive correlations for most cultivars. Effects of iron treatments on storage duration were also analyzed for both visual and digital systems. There were significant differences among MNDVI values for plantlets stored on medium with standard MS iron alone (EDTA chelated) and with the addition of sequestrene 138 iron. In general, the MNDVI value of the upper node correlated well with visual ratings and could be used to determine the health of in vitro stored hops.