Abstract: Pearls are popular organic gems formed by biological processes. The price differences between seawater and freshwater cultured pearls create significant market demands for robust methods to differentiate between the two types. This demand is particularly important for pearls exhibiting similar characteristics, such as colour, shape, size, and surface texture, for example, those observed in white "freshwater Akoya pearls" and Akoya pearls¹. In this study, the utilization of Sr isotope composition is proposed as a tool for distinguishing between freshwater and seawater cultured pearls. A single-second laser ablation protocol coupled with a linear regression calibration method² was developed for Sr isotope analysis to differentiate pearl types (i.e., freshwater pearls or seawater pearls) while causing minimum damage with the ablation time being reduced from 30-60 s to only 1 s (Fig. 1). Additionally, a pearl reference material (RM), GIC-P, was developed for micro and in situ Sr isotope analysis. The ablation behavior, involving laser ablation spot sizes, repetition rates, and energy densities required for accurate Sr isotope analysis, was investigated. In general, the single-second pulse LA-MC-ICP-MS analytical method developed in this study minimizes sample consumption and offers identification between seawater pearls and freshwater pearls with quasi "non-destructive" capabilities.With the proposed method and the newly developed RM, it was reported for the first time that seawater cultured pearls consistently exhibit the ⁸⁷Sr/⁸⁶Sr ratio around 0.709 2, consistent with modern seawater values, while freshwater cultured pearls display much higher and variable ⁸⁷Sr/⁸⁶Sr ratios ^3-4. The collected freshwater cultured pearls from the Chinese market yielded values ranging from 0.710 5 to 0.71 23, aligning well with the Sr isotopes composition in freshwater samples along the Yangtze River⁵. The results indicate that the Sr isotope in pearl samples depends largely on the Sr isotope composition of the ambient water environment, and discrimination between seawater cultured pearls and freshwater cultured pearls using the Sr isotopes ratio is indeed a method tracing the cultivation environment, thus being robust and reliable.