Abstract <p>Exploring the genetic landscape and corresponding regulatory mechanisms influencing morphine and related alkaloid production efficiency in the opium poppy (<i>Papaver Somniferum</i> L.) is important for sustainable medicinal production. However, information regarding the corresponding medicinal properties of poppy subspecies remains elusive, and the correlation between agronomic traits and morphine production remains unclear. this study aims to investigate the distinction between two poppy subspecies: Shan Yang (SY) and Lan Tian (LT). Physiological data revealed that SY exhibited greater tolerance to environmental stress and a higher morphine content than LT, especially during the reproductive stage. To investigate genetic and molecular mechanisms underlying different tolerance traits and morphine synthesis efficiencies, a comprehensive comparison of genomic, transcriptomic, and metabolomic data was conducted.The results showing significantly higher single nucleotide polymorphism (SNP) frequency in SY (23.78%) compared to LT (23.69%). The proportions of SKIP (Skipped Exon) and MIR (Multi Intron Retention) were higher in SY than in LT. SY showed significantly higher gene expression related to benzylisoquinoline alkaloid synthesis, especially those involved in the steps from 1,2 dehydroreticulinium to morphine, narcotoline and noscapine, compared to LT. The upregulated expression of phenylpropanoid biosynthesis-related genes contributes to its more resistant agronomical traits. It plays a synergistic role in alkaloid production. The higher morphine content in SY resulted from an integrated effect controlled at different levels. Our findings offer new insights into the molecular mechanisms of morphine synthesis and present valuable gene resources for improving poppy cultivars with higher morphine content.</p> Graphical abstract