Background <p>High-altitude hypoxia (HAH) can cause adverse reactions, such as tinnitus and barotrauma, but the role of mitotic catastrophe (MC) in HAH remains unreported. This study investigated MC-associated biomarkers in HAH.</p> Methods <p>HAH-related datasets and MC-related genes (MC-RGs) were retrieved from public databases. Differentially expressed genes (DEGs) between the HAH and control groups were screened, and overlapping genes between DEGs and MC-RGs were defined as candidates. Biomarkers were identified using three machine learning methods, receiver operating characteristic curve analysis, and expression validation. A nomogram was constructed for diagnostic assessment. Functional enrichment, immune infiltration, regulatory network, and drug prediction analyses were performed, with biomarker expression verified by reverse transcription quantitative polymerase chain reaction (RT-qPCR).</p> Results <p><i>DIAPH1</i> and <i>MDM4</i> were confirmed as MC-associated biomarkers in HAH, and the nomogram displayed excellent diagnostic ability (area under the curve = 0.929). These biomarkers were coenriched in pathways such as oxidative phosphorylation. <i>DIAPH1</i> was positively correlated with the infiltration of resting NK cells, whereas <i>MDM4</i> was negatively correlated with that of M2 macrophages. A transcription factor–mRNA–miRNA network was established, with <i>USF2</i> and <i>GATA2</i> jointly targeting both biomarkers. Drugs such as rescinnamine targeted <i>MDM4</i>. RT-qPCR verified that <i>USF2</i> and <i>GATA2</i> were significantly downregulated in HAH (both <i>P</i> &lt; 0.01), consistent with the bioinformatics results.</p> Conclusions <p><i>DIAPH1</i> and <i>MDM4</i> are valid MC-associated biomarkers for HAH. This study provides novel insights into HAH prevention and treatment.</p>