Strawberry is the most cultivated berry fruit globally and it is really appreciated by consumers because of its characteristics, mainly bioactive compounds with antioxidant properties. During the breeding process, it is important to assess the quality characteristics of the fruits for a better selection of the material, but the conventional approaches involve long and destructive lab techniques. Near infrared spectroscopy (NIR) could be considered a valid alternative for speeding up the breeding process and is not destructive. In this study, a total of 216 strawberry fruits belonging to four different cultivars have been collected and analyzed with conventional lab analysis and NIR spectroscopy. In detail, soluble solid content, acidity, vitamin C, anthocyanin, and phenolic acid have been determined. Partial least squares discriminant analysis (PLS-DA) models have been developed to classify strawberry fruits belonging to the four genotypes according to their quality and nutritional properties. NIR spectroscopy could be considered a valid non-destructive phenotyping method for monitoring the nutritional parameters of the fruit and ensuring the fruit quality, speeding up the breeding program.