The present invention provides compositions, kits, and methods for detecting polynucleotide sequence differences. The method involves amplifying a polynucleotide in the presence of a labeled nucleotide whose incorporation into the amplified product can indicate the presence of a sequence difference within the polynucleotide template. The invention is particularly useful for differentiating two or more closely related polynucleotide sequences, for example, in determining which allele or alleles of a multiallelic organism are present in a target polynucleotide.