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Background: In situ hybridization (ISH) is a general molecular biological technique used to determine the spatiotemporal expression of genes in many species. In the past few years, numerous ISH protocols have been established in many species. Turnip (Brassica rapa var. rapa) is an important crop in the world, especially in the Plateau area of China, and is a traditional Tibetan medicine. However, ISH protocol in turnip has not been established. Results: We explored and established an optimal workflow for mRNA ISH system for turnip which has been evaluated using BrrCLV3 and BrrWUSa. The optimal methods include: (1) fixation method, (2) protease K pretreatment time, (3) probe length and concentration, (4) washing temperature. We also provide advice on weakening background and improving the efficiency of RNA transcription in vitro. The expression of the BrrCLV3 gene in turnip was detected by the optimized system, and the applicability of the system was confirmed by using BrrWUSa. Conclusions: In this study, we established and optimized the mRNA ISH system for turnip. We explored and found that (1) FAA fixative was the optimized fixation method, (2) 30 min was the optimized protease K pretreatment time, (3) 100 bp, 100 ng/ml probe had good hybridization signal, (4) the optimized washing temperature was 52 °C. It provides a powerful method to locate mRNA in the tissue, which can study the expression and function of turnip's genes. As such, it has considerable advantages in terms of time and cost. [ABSTRACT FROM AUTHOR]