The Waxy (Wx) gene controls the synthesis of amylose, the key determinant of rice cooking and eating qualities. To date, several Wx alleles and their effects on rice quality have been extensively elucidated. However, studies on the diversity of Wx in Oryza sativa L. landrace "Guizhou HE", which plays important roles in local people's daily lives, have been limited. Here, we investigated polymorphisms in Wx alleles and their correlations with the apparent amylose content (AAC) and gel consistency (GC) in "Guizhou HE". Genotype identification and sequencing data showed that all the glutinous "Guizhou HE" varieties contained a wx allele having a 23-bp duplication in exon 2. At the Wx locus, six (CT) n microsatellite alleles, having n = 17, 18, 19, 20, 21, and 24, were found. Among them, (CT) 20 was the most frequent, and (CT) 21 was reported for the first time. The AAC in glutinous "Guizhou HE" ranged from 0.14% to 5.15%, and the GC ranged from 82.5 mm to 113.5 mm. No significant differences in AAC and GC were found among the (CT) n alleles. In addition, Wx g1 , Wx g4 , and Wx t1 alleles were also identified in non-glutinous "Guizhou HE". In conclusion, our results confirmed the existence of various Wx alleles in "Guizhou HE", and they may serve as excellent resources for genetic diversity determination and rice quality analysis.