The goal of the research was to obtain data for the calibration of portable NIR devices (in the near-infrared region) for fresh and wilted alfalfa Morava and Holyna varieties. The trial took place in two locations in five consecutive years (2018–2022). A total of 108 alfalfa samples from chemical analysis and 1080 samples from the portable AgriNIR spectroscope were included in the statistical evaluation. Chemical analysis showed higher contents of dry matter (by 9 g/kg dry matter), acid detergent fiber (ADF) (by 10.2 g/kg dry matter) and ash (2.2 g/kg dry matter) and lower proteins (by 11.2 g/kg dry matter). In fresh alfalfa, chemical analysis showed lower proteins and higher ADF than NIR spectroscopy. In wilted alfalfa, chemical analysis showed lower proteins and higher neutral detergent fiber and ash content than NIR spectroscopy. The difference between the varieties was only in the ash content of the Morava variety, it was higher in the chemical analysis than the NIRS method.