Carotenoids play an essential role in human health and they also affect the perception of taste and flavour that influence consumer preference. Carrot is one of the most important and consumed vegetables in the world, and a critical source of α- and β-carotene accumulated in root tissues. The carotenoid content of carrots is mainly influenced by cultivar and growing sites. Commonly grown carrot cultivars (hybrids) were obtained from Begeč location (Vojvodina, Serbia): Maestro, Bolero, Natuna and Naval. The objectives of this study were to determine: 1) the carotenoid profile of the carrot cultivars by using the Raman spectroscopy and spectrophotometry, 2) are there any differences among selected cultivars regarding carotenoid content. Raman spectra of the root of four carrot cultivars exhibited three dominant carotenoid signals, mainly related to α- and β-carotene, in the three distinct regions: from 1509-1517 cm^-1, 1146-1157 cm^-1 and 998-1009 cm^-1. According to the PCA analysis of the Raman spectra it is indicated that the first and second principal components are responsible for 94.06% of the total data variance and it suggested the existence of two groups of objects along PC1 axis. The loading plots showed variables with the highest positive and negative contribution along PC1 and PC2, indicating the differences between Maestro and Bolero on the one side and Natuna and Naval on the other, that are mainly based on carotenoid content and in the lower extend in carbohydrates. Raman spectroscopy was applied as a fast tool for the chemical evaluation which provided information about the carrot cultivars differences, mainly contributed to carotenoid content. Spectrophotometric analysis revealed that total carotenoid content was in range from 24.14 to 31.12 μg/g of dry mass (Naval and Bolero).