Objectives: To provide insights for breeding high-quality rice varieties, we analyzed local rice (Oryza sativa L.) germplasm from Yunnan Province, China, focusing on the relationships among Waxy gene alleles, indica–japonica differentiation, and amylose content (AC). Methods: We examined 201 local rice accessions. Two functional molecular markers for the Waxy gene were used to detect four alleles (Wx^a, Wx^b, Wxⁱⁿ, Wx^mw). Additionally, 33 InDel markers were employed to classify indica–japonica attributes, and AC was measured according to GB/T 15683-2008. Results: We detected 175 accessions with Wx^a, 20 with Wx^b, 4 with Wxⁱⁿ, and 2 with Wx^mw, indicating Wx^a dominance and a diverse genetic basis at the Waxy locus. Indica–japonica classification identified 180 indica-type, 19 japonica-type, and 2 intermediate-type accessions, confirming predominant indica differentiation in Yunnan rice. Integrating Waxy allele detection, indica–japonica attributes, and AC showed that Wx^a occurred primarily in indica rice with higher AC (mean 22.55%), comparable to Wxⁱⁿ (mean 24.33%); Wx^b was mainly found in japonica rice with lower AC (mean 13.46%), similar to Wx^mw (mean 15.65%). Conclusions: Local Yunnan rice exhibits Wx^a predominance at the Waxy locus and clear indica differentiation. The observed associations between Waxy alleles, subspecies attributes, and AC provide useful references for marker-assisted breeding of premium rice and for exploiting indica–japonica heterosis.