<b><i>Background:</i></b> Microsatellite instability (MSI) in non-small cell lung cancer (NSCLC) is uncommon; however, most studies refer to European and Asian populations. There are currently no data on MSI frequency in highly admixed populations, such as the one represented by Brazilian NSCLC patients. <b><i>Aim:</i></b> This study aimed to evaluate the frequency of MSI in Brazilian NSCLC patients. <b><i>Methods:</i></b> We evaluated 526 patients diagnosed with NSCLC at the Barretos Cancer Hospital (Brazil). The molecular MSI evaluation was performed using a hexa-plex marker panel by polymerase chain reaction followed by fragment analysis. The mutation profile of MSI-positive cases was performed using next-generation sequencing. <b><i>Results:</i></b> Only 1 patient was MSI positive (0.19%). This patient was a female, white, and active smoker, and she was diagnosed with clinical stage IV lung adenocarcinoma at 75 years old. The molecular profile exhibited 4 <i>Tumor Protein p53 (TP53)</i> mutations and the absence of actionable mutations in the <i>Epidermal Growth Factor Receptor (EGFR)</i>, <i>Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS),</i> or <i>V-Raf Murine Sarcoma Viral Oncogene Homolog B1 (BRAF)</i> genes. <b><i>Conclusions:</i></b> The frequency of MSI in Brazilian NSCLC patients is equally rare, a finding that is consistent with the current literature based on other populations such as Europeans, North Americans, and Asians.