Nakaseomyces glabratus is a medically important fungal pathogen responsible for various opportunistic, life-threatening, and fatal infections, mainly among immunodepressed patients worldwide. Herein, genotypes identified in Central Poland by multilocus sequence typing (MLST) are presented. Along with the genotyping, drug susceptibility was performed. The research was conducted on 30 non-redundant clinical strains, and 15 distinct sequence types (STs) were identified, including three novel STs: ST212, ST213, and ST214. The most prevalent sequence types were ST3, ST6, and ST10. Antifungal susceptibility testing revealed varied resistance rates to azoles, with fluconazole susceptibility at 16.7% and high susceptibility to amphotericin B. No correlation between ST and antifungals MIC were found. The study findings highlight the genetic diversity of N. glabratus in Central Poland and the role of surveillance and research to elucidate antifungals resistance and molecular epidemiology of N. glabratus.