Background: Patients with Down syndrome (DS) are at increased risk for infections and autoimmune disorders. Although several immunological abnormalities were previously found, differences in T cell receptor repertoire have never been shown. Thus we compared the T cell receptor gamma (TRG) repertoire in DS and non-syndromic pediatric patients by next-generation sequencing, in addition to other immunological markers. Methods: Genomic DNA was extracted from thymuses of pediatric patients who underwent heart surgery, where six were with DS and six were non-syndromic patients. Peripheral blood counts, T cell subpopulations, thymus TCR excision circles (TRECs), spectratyping, and next-generation sequencing for TRG were analyzed. Results: The mean age of the patients was 7 months and the mean lymphocyte count was slightly lower in patients with DS, whereas thymus TREC results were similar to non-syndromic patients (p = 0.197). The TRG repertoire analysis showed that patients with DS had a significantly larger number of unique TRG sequences, together with decreased clonal expansion. Lastly, the V and J gene usages in the thymus were similar in DS and non-syndromic patients. Conclusions: Patients with DS showed increased TRG repertoire diversity with decreased clonal expansion compared to non-syndromic patients. Impact: Alterations in T cell receptor gamma repertoire were found in patients with Down syndrome using next-generation sequencing (NGS) technique. Patients showed increased repertoire diversity and decreased clonal expansion compared to controls.These findings add to previous reports on abnormalities of other immune system components in patients with Down syndrome. NGS technique may point out differences not seen by previous methods.Repertoire abnormalities may contribute to those patients’ predisposition to infections and autoimmune diseases.