Horseweed (Conyza canadensis), an invasive alien weed, is one of the main weeds in orchards in China. Although glyphosate has been used for control of horseweed and many other weeds in orchards for more than 25 years in China, a case of glyphosate-resistant horseweed has not been identified in orchard in China so far despite glyphosate-resistant horseweed cases have been reported in some other countries. Seeds of 25 horseweed populations were collected from different orchards with different glyphosate application history. Potted seedlings with 11-13-leaf growth stage were treated with glyphosate at 0.035, 0.07, 0.14, 0.28, 0.56, 1.12, 2.24, 4.48, and 8.96 kg a.i. ha-1. The dosage dependence response curve of each population was constructed with Log-logistic dose response regression equations. The ED50 value of each population was calculated and compared with the susceptible population from China. Different populations had different relative glyphosate-resistant levels which increased with the number of years of glyphosate application. Two populations with the highest resistance levels, 8.28 and 7.95 times, were found in Ningbo, Zhejiang Province, China, where glyphosate was used for weed control in orchards twice each year for 15 yr. The two resistant populations accumulated approximately two to four times less shikimic acid than the two susceptible populations 48 h after glyphosate application. Horseweed (Conyza canadensis), an invasive alien weed, is one of the main weeds in orchards in China. Although glyphosate has been used for control of horseweed and many other other weeds in orchards for more than 25 years in China, a case of glyphosate- resistant horseweed has not been identified in orchard in China so far despite glyphosate-resistant horseweed cases have been reported in some other countries. Seeds of 25 horseweed populations were collected from different orchards with different glyphosate application history. Potted seedlings with 11-13-leaf growth stages were treated with glyphosate at 0.035, 0.07, 0.14, 0.28, 0.56, 1.12, 2.24, 4.48, and 8.96 kg ai ha-1. The dosage dependence response curve of each population was constructed with Log- logistic dose response regression equations. The ED50 value of each population was calculated and compared with the susceptible population from China. Different populations had different relative glyphosate-resistant levels which increased w Two the number of years of glyphosate application. Two populations with the highest resistance levels, 8.28 and 7.95 times, were found in Ningbo, Zhejiang Province, China, where glyphosate was used for weed control in orchards twice each year for 15 yr. The two resistant populations accumulated about two to four times less shikimic acid than the two susceptible populations 48 h after glyphosate application.