Amylose content (AC) is the major indicator of rice eating and cooking quality (ECQ). Its synthesis in rice endosperm is mainly regulated by the protein, granule-bound starch synthase 1, which is encoded by the waxy gene (Os06g0133000, LOC_Os06g04200). The diversity of AC is largely attributable to the allelic variation at the Wx locus and the development of effective and accurate functional molecular markers to target rice variant alleles is crucial in the breeding strategy. In the present study, we developed six pairs of penta-primer amplification refractory mutation system (PARMS) markers to distinguish between Wxlv, Wxa, Wxin, Wxb, Wxmp, and Wxop,hp. These markers were successfully used to screen the genotype of large assets of genetic resources including 98 wild accessions, 55 cultivars, and 22 parental lines. Our results showed that Wxb in a low AC type was predominant in Guangxi cultivated rice as a result of cultural preference, while Wxlv in the wild accessions. Moreover, our findings surprisingly revealed the presence of Wxb in wild accession, which is a new outcome that may contribute to understanding the origin, selection and domestication processes of rice. These functional markers could be effectively used in marker-assisted breeding to improve selection efficiency of cultivars with desired AC in the early generation.