We have adapted the mutation detection technology used in Targeting Induced Local Lesions in Genomes (TILLING) to the discovery of polymorphisms in natural populations. The genomic DNA of a queried individual is mixed with a reference DNA and used to amplify a target 1-kbp region of DNA with asymmetrically labeled fluorescent primers. After heating and annealing, heteroduplexes are nicked at mismatched sites by the endonuclease CEL I and cut strands are visualized using Li-cor gel analyzers. Putative polymorphisms detected in one fluorescence channel can be verified by appearance of the opposite cut strand in the other channel. We demonstrated the efficiency of this technology, called Ecotilling, by the discovery in 150+ individuals of 55 haplotypes in five genes, ranging from sequences differing by a single nucleotide polymorphism to those representing complex haplotypes. The discovered polymorphisms were confirmed by sequencing and included base-pair changes, small insertions and deletions, and variation in microsatellite repeat number. Ecotilling allows the rapid detection of variation in many individuals and is cost effective because only one individual for each haplotype needs to be sequenced. The technology is applicable to any organism including those that are heterozygous and polyploid.