A blueberry (Vaccinium sp.) germplasm collection was screened for allelic variation in TERMINAL FLOWER 1 (TFL1) that could be used in breeding flowering or architecture traits. TFL1 has been found to repress the transition from vegetative to reproductive growth in diverse plant species, with mutations leading to altered flowering and form. The VcTFL1 gene sequence was determined from the draft genome sequence of diploid V. corymbosum line W8520. VcTFL1 is a member of a PEBP gene family and it could be distinguished from its family members by sequence comparison with PEBP family genes from other plants. High-resolution DNA melting analysis of 160 Vaccinium accessions detected VcTFL1 exons that differed in sequence from the W8520 control. DNA sequence analysis confirmed the presence of single nucleotide polymorphisms (SNPs) and identified haplotypes of tetraploid accessions. A total of 18 SNP sites were detected in VcTFL1 coding sequences of the Vaccinium germplasm screened. A SNP causing an alanine-to-valine change in exon 4 (A159V) was determined by multiple bioinformatic tools to be deleterious to VcTFL1 function. A diploid V. corymbosum accession heterozygous for the VcTFL1 mutation was identified as a candidate for breeding novel traits for blueberry.