Assessing species’ range-wide cytoplasmic diversity provides valuable insights regarding their dispersal and adaptive potential in a changing environment. Transcontinental chloroplast (cpDNA) and mitochondrial DNA (mtDNA) population structures were compared to identify putative ancestral and new cytoplasmic genome assemblages in black spruce (Picea mariana), a North American boreal conifer. Mean within-population diversity and allelic richness for cpSSR markers were 0.80 and 4.21, respectively, and diminished westward. Population differentiation based on GST was lower for cpDNA than for mtDNA (GST = 0.104 and 0.645, respectively) but appeared comparable when estimated using Jost differentiation index (D = 0.459 and 0.537, respectively). Further analyses resulted in the delineation of at least three genetically distinct cpDNA lineages partially congruent with those inferred from mtDNA data, which roughly corresponded to western, central and eastern Canada. Additionally, the patterns of variation in Alaska for both cpDNA and mtDNA markers suggested that black spruce survived the last glacial maximum in this northern region. The range-wide comparison of the geographic extent of cytoplasmic DNA lineages revealed that extensive pollen gene flow between ancestral lineages occurred preferentially from west to east during the postglacial expansion of the species, while seed-mediated gene flow remained geographically restricted. This differential gene flow promoted intraspecific cytoplasmic capture that generated new assemblages of cpDNA and mtDNA genomes during the Holocene. Hence, black spruce postglacial colonization unexpectedly resulted in an increase in genetic diversity with possible adaptive consequences.