Crayfish (Procambarus clarkii) is one of the most important economic crustacean species in China. In 2020, the area farmed P. clarkii was 0.14 million hectares, with production exceeding 2.39 million tons, which ranked the 6th among freshwater aquaculture species in China (the top 5 are all large freshwater fish species). As an important and popular freshwater aquatic product in China, the food safety of P. clarkii is widely concerned by consumers. Trace element level is an important index to evaluate food safety. In order to investigate the concentrations of trace elements in rice-crayfish and its food safety, 24 trace elements in 156 samples of P. clarkii cultured in rice fields in Hubei, Hunan and Anhui were detected by inductively coupled plasma mass spectrometry (ICP-MS). Crayfish cultured in rice fields of Hubei, Hunan and Anhui three provinces were collected from May to June in 2021. A total of 156 samples were collected, including 55 from Hubei province, 56 from Hunan province and 45 from Anhui province. The pollution levels and potential health risks were estimated by pollution indices, exposure assessment and non-carcinogenic risk assessment method. Twenty trace elements had been detected in P. clarkii samples, and Li, Be, Tl and U were not detected. The contents of 17 trace elements with a detection rate of more than 50% were in order from high to low: Zn>Fe>Cu>Rb>Al>Mn>Sr>Ba>Se>As>Mo>Cs>Ag>V≈Cr>Co>Ga. The average content of Zn was the highest (5.384±4.650 mg/kg), followed by Fe [(3.153±1.898) mg/kg] and Cu [(2.459±0.919) mg/kg]. Except Rb and Al, the content of essential trace elements in muscle of P. clarkii was higher than that of harmful trace elements. Sixteen of the 24 trace elements in the rice-crayfish samples had significant differences between Anhui and other two provinces, and 5 of them had significant differences between the three regions. The contents of 6 heavy metals (Cu, Zn, As, Cr, Pb and Cd) in crayfish samples were lower than those reported in previous studies. The possible reason was monitoring time, location and other factors, especially the sampling site. Some trace elements in rice-crayfish muscle showed significant regional differences by phylogenetic relation analysis, which indicated that the enrichment of trace elements in aquatic products was related to the growing environment. The pollution indices of five toxic heavy metals (As, Cu, Pb, Cd and Cr) in rice-crayfish samples from three provinces were all lower than 0.2 and at safe levels. The estimated daily intakes(EDI) of 7 elements (Cu, Zn, Cr, Al, As, Pb and Cd) were 1.213, 2.656, 0.003, 0.899, 0.030, 0.0005 and 0.00005 μg/(kg·d), respectively. The intake of the 7 elements accounted for 0.125%-10.862% of the ADI values of adults. Consumption of Cu, Zn, Cr, Al, As, Pb and Cd from P. clarkii in the survey area was associated with low health risk. Except for Fe element (THQ=1.111), the THQ values of other 20 elements were all lower than 1, and the total target hazard quotient (TTHQ) was 3.672. TTHQ was lower than 10, which indicated that the P. clarkii muscle samples analyzed in this study had no toxic risk effect. However, Fe intake is of concern.