SARS-CoV-2 is constantly evolving, leading to new variants. We analysed data from 4400 SARS-CoV-2-positive samples in order to pursue epidemiological variant surveillance and to evaluate their impact on public health in Italy in the period of April–December 2021. The main circulating strain (76.2%) was the Delta variant, followed by the Alpha (13.3%), the Omicron (5.3%), and the Gamma variants (2.9%). The B.1.1 lineages, Eta, Beta, Iota, Mu, and Kappa variants, represented around 1% of cases. There were 48.2% of subjects who had not been vaccinated, and they had a lower median age compared to the vaccinated subjects (47 vs. 61 years). An increasing number of infections in the vaccinated subjects were observed over time, with the highest proportion in November (85.2%). The variants correlated with clinical status; the largest proportion of symptomatic patients (59.6%) was observed with the Delta variant, while subjects harbouring the Gamma variant showed the highest proportion of asymptomatic infection (21.6%), albeit also deaths (5.4%). The Omicron variant was only found in the vaccinated subjects, of which 47% had been hospitalised. The diffusivity and pathogenicity associated with the different SARS-CoV-2 variants are likely to have relevant public health implications, both at the national and international levels. Our study provides data on the rapid changes in the epidemiological landscape of the SARS-CoV-2 variants in Italy.