We investigated the utility of association mapping to dissect the genetic basis of naturally occurring variation in bud phenology in European aspen (Populus tremula). With this aim, we surveyed nucleotide polymorphism in 13 fragments spanning an 80-kb region surrounding the phytochrome B2 (phyB2) locus. Although polymorphism varies substantially across the phyB2 region, we detected no signs for deviations from neutral expectations. We also identified a total of 41 single nucleotide polymorphisms (SNPs) that were subsequently scored in a mapping population consisting of 120 trees. We identified two nonsynonymous SNPs in the phytochrome B2 gene that were independently associated with variation in the timing of bud set and that explained between 1.5 and 5% of the observed phenotypic variation in bud set. Earlier studies have shown that the frequencies of both these SNPs vary clinally with latitude. Linkage disequilibrium across the region was low, suggesting that the SNPs we identified are strong candidates for being causally linked to variation in bud set in our mapping populations. One of the SNPs (T608N) is located in the “hinge region,” close to the chromophore binding site of the phyB2 protein. The other SNP (L1078P) is located in a region supposed to mediate downstream signaling from the phyB2 locus. The lack of population structure, combined with low levels of linkage disequilibrium, suggests that association mapping is a fruitful method for dissecting naturally occurring variation in Populus tremula.