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Understanding the process of signal communication between pollen and stigma is of significant importance for plant sexual reproduction. In the case of inter‐subgeneric hybridization in water lily, a pre‐fertilization hybridization barrier exists, the regulatory mechanism of which remains unclear. In this study, we conducted hormone and transcriptome analyses of unpollinated stigmas (Mock), self‐pollinated stigmas (SP), cross‐pollinated stigmas within the same subgenus (CP), and inter‐subgenus cross‐pollination stigmas (ISCP) in water lily to elucidate the formation mechanism of the inter‐subgeneric hybridization barrier. Our results indicated that the lack of abscisic acid (ABA) and brassinolide (BR) in ISCP stigmas are key factors contributing to the formation of the inter‐subgeneric hybridization barrier in water lily. Exogenous application of ABA and BR can help overcome the barrier between inter‐subgeneric water lily crosses. Through transcriptome analysis, we identified nine candidate genes involved in regulating the inter‐subgeneric hybridization barrier in water lily. In addition, we further demonstrated the importance of the NCED2‐mediated ABA synthesis pathway in the pollination process through AS‐ODN technology. Our study confirms that ABA and BR are critical for breaking the inter‐subgeneric hybridization barrier. The identification of the nine candidate genes provides important clues for further research on the hybridization recognition mechanism in water lily. [ABSTRACT FROM AUTHOR]