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Sixteen cultivars (three resistant species and thirteen susceptible cultivars to cochineal insects) were included in the experiment to evaluate their nutritional and chemical compositions and to determine the in vitro digestibility of cladodes of the Opuntia species. Cultivars showed highly significant (p < 0.001) differences in their chemical composition. The cultivars’ content of ash, crude protein (CP), dry matter (DM), organic matter (OM), in vitro dry matter digestibility (IVDMD), in vitro organic matter digestibility (IVOMD), acid detergent fiber (ADF), neutral detergent fiber (NDF) and Cell (cellulose) varied, with ranges of 8.18–22.75%, 3.19–10.40%, 86.40–91.88%, 77.45–91.82%, 68.25–87.3%, 55.81–78.95%, 14.88–26.15%, 29.66–67.05% and 10.99–19.92%, respectively. The ash content showed a significant highly negative correlation with OM (r = −1.00; p < 0.001), IVDMD (r = −0.603; p < 0.001) and DOMD (r = −0.904; p < 0.001), respectively. The Garao, Aloqa and O. robusta var. X11 cultivars showed the highest similarities, as they are found close to the first branch of the dendrogram. In total, 90% of the variation among the nutritional traits was attributed to the first four PCs, with 55.97% to the first two principal components (PCs). The nutritional composition of the resistant species was greater than or equal to the susceptible ones.