<b><i>Background:</i></b> Mixed adenoneuroendocrine carcinomas (MANECs) of the gastrointestinal tract are rare neoplasms characterized by coexisting exocrine and neuroendocrine neoplastic components. MANECs' histogenetic classification and molecular characterization remain unclear, significantly affecting the identification of innovative therapeutic options for these tumors. <b><i>Methods:</i></b> The exocrine and neuroendocrine components of 6 gastrointestinal MANECs were microdissected and subjected to the simultaneous mutation assessment in selected regions of 54 cancer-associated genes using Ion Torrent semiconductor-based next-generation sequencing. Sanger sequencing and immunohistochemistry were used as validation of the mutational status. <b><i>Results:</i></b> A total of 20 driver gene somatic mutations were observed among the 12 neoplastic components investigated. In 11 of 12 (91.7%) samples, at least one mutation was detected; 7 samples (58.3%) were found to have multiple mutations.<i> TP53</i> gene mutations were the most frequent genetic alterations observed in the series, occurring in 11/12 samples (91.7%). Somatic mutations in other genes were detected at lower frequencies: <i>ATM</i>, <i>CTNNB1</i>, <i>ERBB4</i>, <i>JAK3</i>, <i>KDR</i>, <i>KRAS</i>, <i>RB1</i>. <b><i>Conclusions:</i></b> Five of the 6 MANECs presented an overlapping mutational profile in both components, suggesting a monoclonal origin of the two MANEC components.