The determination of strawberry fruit quality through the traditional destructive lab techniques has some limitations related to the amplitude of the samples, the timing and the applicability along all phases of the supply chain. The aim of this study was to determine the main qualitative characteristics through traditional lab destructive techniques and Near Infrared Spectroscopy (NIR) in fruits of five strawberry genotypes. Principal Component Analysis (PCA) was applied to search for spectral differences among all the collected samples. A Partial Least Squares regression (PLS) technique was computed in order to predict the quality parameters of interest. The PLS model for the soluble solids content prediction was the best performing—in fact, it is a robust and reliable model and the validation values suggested possibilities for its use in quality applications. A suitable PLS model is also obtained for the firmness prediction—the validation values tend to worsen slightly but can still be accepted in screening applications. NIR spectroscopy represents an important alternative to destructive techniques, using the infrared region of the electromagnetic spectrum to investigate in a non-destructive way the chemical–physical properties of the samples, finding remarkable applications in the agro-food market.