Apparent amylose content (AAC) is a key determinant of eating and cooking quality in rice and it is mainly controlled by the Wx gene which encodes a granule-bound starch synthase (GBSS). In this study, sixteen single-segment substitution lines harboring the Wx gene from 16 different donors and their recipient HJX74 were used to detect the naturally occurring allelic variation at the Wx locus. The AAC in the materials varied widely and could be grouped into glutinous, low, intermediate, and two high AAC sub-classes, high I (24.36–25.20%) and high II (25.81–26.19%), under different experimental environments, which showed a positive correlation with the enzymatic activity of GBSS. One insertion/deletion (InDel) and three single nucleotide polymorphisms in the Wx gene were detected and their combinations resulted in the variation of five classes of AAC. Based on the results of AAC phenotypes, GBSS activities and cDNA sequences, five Wx alleles, wx, Wx t, Wx g1, Wx g2, and Wx g3, were identified, two of which, Wx g2 and Wx g3, are separated for the first time in this study. Under different cropping seasons, the AAC differed significantly for the Wx t and Wx g1 alleles, with higher AAC in the fall season than in the spring season, but did not differ significantly for the wx, Wx g2, and Wx g3 alleles. In conclusion, the present results might contribute to our understanding of the naturally occurring allelic variation at the Wx locus and will facilitate the improvement of rice quality by marker-assisted selection.