BACKGROUND: tsRNAs are novel small non-coding RNAs that play important regulatory roles in gene expression, translation, transcription, and epigenetic modification through proteins or mRNAs and may be therapeutic targets for certain diseases. The etiology of non-syndromic cleft palate-only is complex and the pathogenesis is poorly understood, non-coding RNAs play important roles in its development. METHODS: The tsRNAs of patients with simple cleft palate were compared with healthy individuals using small RNA microarray, bioinformatic analysis, quantitative real-time transcription polymerase chain reaction, and the effects measured using immunohistochemical staining. RESULTS: Seventy-nine tsRNAs were upregulated and fifty-four tsRNAs were downregulated in patients with simple cleft palate compared with healthy individuals, among which the expression of 5'tiRNA-35-GlyTCC-3 and 5'tiRNA-33-CysGCA-11 was markedly different and was involved in key signaling pathways related to the development of the palate, such as the cell cycle, cAMP signaling pathway, BMP signal transduction, folate biosynthesis, and other key signaling pathways that determine anatomical structure occurrence, regulate gene expression during development, influence epigenetics, and other biological processes, its target genes include BMP6, CUL1 and SPR. CONCLUSION: 5'tiRNA-35-GlyTCC-3 and 5'tiRNA-33-CysGCA-11 are closely associated with non-syndromic cleft palate development and are expected to be potential new targets for diagnosis and treatment.