Rice blast causes huge losses of rice and is the limiting factor in rice production. Host resistance improvement using marker-assisted selection (MAS) is considered to be the most economic and effective way to overcome rice blast. In this study, an allele mining strategy was used to find variation of the Pi2 blast-resistance gene from different rice varieties. In some varieties, there was an out-of-frame deletion of 3 base-pairs (bp) at the 1543–1545 site of the Pi2 gene (the “deletion” allele). Deletion of these 3 bp causes the loss of cysteine–arginine and their replacement with glycine in the protein product. As a consequence the predicted protein structure is changed, especially in the LRR region which is important to plant blast resistance. Plants with the deletion allele showed poor resistance compared to those with the non-deletion variant. Haplotype analysis showed that the deletion of these 3 bp was common only in indica rice, and the rice varieties with 3 bp deletion perform poor rice blast resistance, which indicates that the deletion allele variant of Pi2 occurred recently and was artificially selected. We developed a functional molecular marker Pi2-1, spanning base pairs 1543–1545 of the Pi2 gene, which clearly distinguished between rice varieties with good or inferior blast resistance. Finally, Pi2-1 was used for rice blast MAS in the Huazhan/R900 F₂ segregation population, and excellent plants with good rice blast resistance were selected.