Mutations have driven the evolution and development of new variants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with potential implications for increased transmissibility, disease severity and vaccine escape among others. Genome sequencing is a technique that allows scientists to read the genetic code of an organism and has become a powerful tool for studying emerging infectious diseases. Here, we conducted a cross-sectional study in selected districts of the Eastern Province of Zambia, from November 2021 to February 2022. We analyzed SARS-CoV-2 samples ( = 76) using high-throughput sequencing. A total of 4097 mutations were identified in 69 SARS-CoV-2 genomes with 47% (1925/4097) of the mutations occurring in the spike protein. We identified 83 unique amino acid mutations in the spike protein of the seven Omicron sublineages (BA.1, BA.1.1, BA.1.14, BA.1.18, BA.1.21, BA.2, BA.2.23 and XT). Of these, 43.4% (36/83) were present in the receptor binding domain, while 14.5% (12/83) were in the receptor binding motif. While we identified a potential recombinant XT strain, the highly transmissible BA.2 sublineage was more predominant (40.8%). We observed the substitution of other variants with the Omicron strain in the Eastern Province. This work shows the importance of pandemic preparedness and the need to monitor disease in the general population.