Tracing the appearance and evolution of virus variants is essential in the management of the COVID-19 pandemic. Here, we focus on SARS-CoV-2 spread in Italian patients by using viral sequences deposited in public databases and a tracing procedure which is used to monitor the evolution of the pandemic and detect the spreading, within the infected population of emergent sub-clades with a potential positive selection. Analyses of a collection of monthly samples focused on Italy highlighted the appearance and evolution of all the main viral sub-trees emerging at the end of the first year of the pandemic. It also identified additional expanding subpopulations which spread during the second year (i.e., 2021). Three-dimensional (3D) modelling of the main amino acid changes in mutated viral proteins, including ORF1ab (nsp3, nsp4, 2’-o-ribose methyltransferase, nsp6, helicase, nsp12 [RdRp]), N, ORF3a, ORF8, and spike proteins, shows the potential of the analysed structural variations to result in epistatic modulation and positive/negative selection pressure. These analyzes will be of importance to the early identification of emerging clades, which can develop into new “variants of concern” (i.e., VOC). These analyses and settings will also help SARS-CoV-2 coronet genomic centers in other countries to trace emerging worldwide variants.