BackgroundHER2-positive breast cancer occurs in 15–20% of breast cancer patients and is characterized by poor prognosis. Trastuzumab is considered the key drug for treatment of HER2-positive breast cancer patients. It improves patient survival; however, resistance to trastuzumab remains a challenge in HER2-positive breast cancer patients. Therefore, the prediction of response to trastuzumab is crucial to choose optimal treatment regimens. The aim of the study was to identify genetic variants that could predict response to anti-HER2-targeted therapy (trastuzumab) using next-generation sequencing.MethodGenetic variants in the hotspot regions of 17 genes were studied in 24 Formalin-Fixed Paraffin-Embedded (FFPE) samples using Ion S5 next-generation sequencing system. FFPE samples were collected from HER2‑positive breast cancer patients previously treated with anti‑HER2‑targeted treatment (Trastuzumab). Patients were divided into two groups; trastuzumab-sensitive group and trastuzumab-resistant group based on their response to targeted therapy.ResultsWe identified 29 genetic variants in nine genes that only occurred in trastuzumab-resistant patients and could be associated with resistance to targeted therapy includingTP53, ATM, RB1, MLH1, SMARCB1, SMO,GNAS,CDH1, andVHL.Four variants out of these 29 variants were repeated in more than one patient; two variants inTP53, one variant inATMgene, and the last variant inRB1gene. In addition, three genes were found to be mutated only in resistant patients;MLH1, SMARCB1 and SMOgenes. Moreover, one novel allele (c.407A > G, p. Gln136Arg) was detected within exon 4 ofTP53gene in one resistant patient.ConclusionNGS sequencing is a useful tool to detect genetic variants that could predict response to trastuzumab therapy.Supplementary InformationThe online version contains supplementary material available at 10.1186/s40246-023-00493-5.