Background: Since the beginning of the pandemic, contact tracing has been one of the most relevant issues to understand SARS-CoV-2 transmission dynamics and, in this context, the analysis of quasispecies may turn out to be a useful tool for outbreak investigations. Analysis of the intra-host single nucleotide variants (iSNVs) found in the nsp2, ORF3, and ORF7 genes of SARS-CoV-2 was conducted in order to correctly identify virus transmission chain among patients hospitalized in Brescia Civic Hospital. Methods: During the period between August and October 2023, 13 nasopharyngeal specimens, collected from patients admitted to Brescia Civic Hospital, were tested for SARS-CoV-2 positivity and molecularly characterized. Firstly, a phylogenetic analysis was performed to evaluate if they were epidemiologically linked and, then, the Beta-binomial method was used to estimate the transmission bottleneck size (Nb) and quantify the number of viral particles transmitted from one individual (donor) to another (recipient). Results: According to the molecular characterization of specimens, we identified two transmission clusters in the cardiology unit: the first cluster concerned patients tested positive for the HV.1/EG.5.1.6 lineage, while the second cluster concerned patients tested positive for the FL.10.1 lineage. Moreover, evaluating the bottleneck size, we were able to solve SARS-CoV-2 transmission chain among infected patients. Conclusion: Our method shows that it is possible to conduct a tracing study using a genomic approach based on iSNVs analysis.