The aims of this study were 1) to determine the variability in the flowering phenology of Scots pine (Pinus sylvestris L.) clones in a seed orchard and 2) to compare the genetic structure and genetic markers (13 isozyme loci and 5 chloroplast and 3 nuclear DNA microsatellite loci) among groups of clones that are differentiated by flowering phenology. Using the timing of male inflorescence development, 57 plus trees represented by their clones in a seed orchard were classified into three phenological groups: early-, intermediate-, and late-flowering. The microsatellites showed no significant differences in the genetic structure of the analyzed phenological groups. However, the frequency of allele 2 at the shikimate dehydrogenase A locus (ShDH A 2) differed significantly between the groups of early- and late-flowering trees and between the groups of intermediate- and late-flowering trees. In addition, a significant difference in the frequencies of the genotype ShDH A 11 was observed between the intermediate- and late-flowering groups. Nei’s genetic distance indicated that the late-flowering group was the most genetically distant among the phenological groups. These results suggest that the ShDH A locus might be considered as isoenzymatic marker that differentiates these flowering groups of Scots pine clones. At several isozyme and DNA loci, the presence of private alleles in each group of pines was observed. However, these alleles cannot serve as markers of Scots pine flowering time because of their low frequencies.