Deviations of the site frequency spectrum of mutations (SFS) from neutral expectations may be caused by natural selection or by demographic processes such as population subdivision or temporal changes in population size. As most widespread temperate and boreal tree species have expanded from glacial refugia in the past 13 000 years, colonization bottlenecks associated with this migration may have left variable demographic signatures among geographic populations corresponding to distance from the refugia. To determine whether the signature of postglacial re-colonization has skewed the SFS in the widely distributed conifer Sitka spruce (Picea sitchensis (Bong.) Carr.), we re-sequenced 153 nuclear genes in six populations from across the species range. We found that while the SFS for the pooled sample produced negative values for Tajima’s D and Fay and Wu’s H, these statistics exhibited strong clinal variation when populations were analysed separately (R2 = 0.84, P = 0.007 for Tajima’s D and R2 = 0.65, P = 0.033 for Fay and Wu’s H). When historical bottlenecks of varying age were simulated using approximate Bayesian computation, distance of populations from the southern range limit explained most of the variation in bottleneck timing among populations (R2 = 0.89, P = 0.003). These data suggest that sequential population bottlenecks during postglacial re-colonization have resulted in diverse among-population signatures within the contemporary SFS in Sitka spruce, with rare variants more common in the south, and medium-frequency variants more common in the north. Our results also emphasize the need to consider sampling strategy and to explore population-specific null demographic models in surveys of nucleotide variation in widely distributed species.