Maize (Zea mays L.) is a promising crop for improving the nutritional quality of foods, because of the presence of broad natural variability for the content of carotenoids. One hundred Italian and nine public lines were screened by PCR to identify the alleles of Hydroxylase3 (HYD3) and Lycopeneepsilon cyclase(LCYE) genes, codifying for key enzymes of the carotenoid pathway and associated with high or low β-carotene contents. Nineteen selected genotypes carrying different alleles were grown in 2011 and/or 2012, and used to obtain F1 seeds. HPLC analysis highlighted the richness in zeaxanthin and β-cryptoxanthin of Italian germplasm (mean values 12.63 and 2.88 mg/kg d.m., respectively). The percentage of positive and significant mid parent heterosis (MPH) and best parent heterosis (BPH) values (51% and 29%) indicated the presence of a high heterotic response for carotenoids. The compounds on the same branch of the pathway were found to be positively correlated; on the contrary, no consistent correlation was observed between the different HYD3 alleles and β-carotene content. Finally, total antioxidant capacity (TAC) was also measured. These lines resulted therefore as interesting raw materials for the market of maize-based foods.