In this paper, three bioassay methods, i.e., relay seeding in agar (RSA), relay seeding in silica (RSS) and seeding in rice root-exudation (SRE), were used to test the allelopathic potential of 8 rice cultivars on the target weed barnyardgrass (Echinochloa crusgalli). The results indicated that RSA was the ideal method for the bioassay, showing the highest efficiency in the evaluation of allelopathic potential. RSS and SRE had a lower efficiency than RSA, but these two methods showed the same tendency in evaluating the allelopathic potential of rice. RSA, the considered best bioassay method in this experiment, was used for 57 allelopathic rice germplasm screening, and 5 of them, i.e., Iguape Cateto, PI312777, Azucena, Taichung Native 1 and IAC25 performed the strongest allelopathic potential in the suppression on barnyardgrass.