Background: Our previous study reported a good efficacy and safety of pyrotinib combined with trastuzumab neoadjuvant treatment in human epidermal growth factor receptor 2 (HER2)-positive breast cancer patients. We further explored the potential biomarkers for the efficacy of pyrotinib combined with trastuzumab neoadjuvant treatment in HER2-positive breast cancer patients. Methods: To date, a total of 96 patients with early-stage breast cancer were enrolled for the neoadjuvant pyrotinib combined with trastuzumab treatment clinical trial (ChiCTR1900022293). By the method of 425 genes next-generation sequencing (NGS), the genomic characteristics of them were evaluated for the potential correlation with postoperative pathological complete response (pCR). Results: Among the cohort of 96 cases, a total of 32 patients have completed the whole therapy as well as final surgery and acquired qualified sequencing analysis report, and 18 of them achieved total pCR. The most frequently mutated driver genes were TP53 (75%), PIK3CA (44%), NBN (9%), RUNX1 (9%), FANCD2 (9%), ATRX (9%), MAP3K1 (9%) and NF1 (9%), respectively. In terms of somatic copy number alterations, the most frequent alterations are gain or amplification of ERBB2 (63%), MYC (22%), CCND1 (19%), CDK12 (16%) and FGF19 (16%), respectively. The median tumor mutation burden (TMB) of the 36 patients was 4.23 mut/Mb (0.00-29.61). Compared with pCR populations, non-pCR populations had significantly higher median TMB (5.29 vs 3.17 mut/Mb, P = 0.025). In addition, the pCR rate of patients with wild-type PIK3CA is significantly higher than that of patients with mutated PIK3CA (88.9% vs 14.3%, P < 0.001). Conclusions: Preliminary results suggested that HER2-positive breast cancer patients with higher TMB and activating mutations in PIK3CA are less likely to benefit from pyrotinib combined with trastuzumab neoadjuvant therapy, which need larger sample size to validate. *Qiyun Shi and Juncheng Xuhong contributed equally. #Co-corresponding author (Dr. Jun Jiang, jcbd@medmail.com.cn; Dr. Xiaowei Qi, qxw9908@foxmail.com). Clinical trial information: ChiCTR1900022293.