We developed a next-generation SARS-CoV-2 sequencing platform and obtained the first SARS-CoV-2 sequences from patients in Croatia at the beginning of the COVID-19 outbreak in the spring of 2020. Integrating the sequencing and the epidemiological data, we show that patients were infected with different SARS-CoV-2 variants belonging to different clades (mostly G and GH). This result confirms that there was widespread virus transmission early in 2020. Interestingly, we identified a unique mutation resulting in a V13I substitution in Nsp5A, the main viral protease, in a patient who had not received antiviral therapy.