Genome features can interact with evolutionary processes and involve in the formation of differentiated regions potentially containing adaptation and speciation loci. However, GC content that can elevate regional mutation rate and is positively correlated with recombination has not been investigated in evolving lineages. Here, we employed 499 genomes of Apis cerana, with a widely distributed Central lineage diverged with its peripheral lineages at both population genetic and phylogenetic timescales, to investigate mutation accumulation and lineage divergence along the speciation continuum. We found differentiated regions are generally with lower recombination and GC compared with the rest of the genome, and with lower divergence (dxy) initially to higher ones at deeper timescale. Higher mutation load in low-GC regions in all A. cerana lineages suggest the important role of restricted recombination instead of selection on differentiated region formation. In addition, most mutations are AT-biased that derived from GC, resulting in lower mutation rate and nucleotide diversity in low-GC regions. AT-biased mutations can be counteracted by GC-biased gene conversion (fixation of GC alleles). While in low-GC regions, we found higher percentage of nearly fixed AT alleles in all peripheral lineages compared with Central lineage, supporting the contribution of AT-biased mutations to lineage divergence. Finally, low-GC regions possess higher proportion of lineage-specific polymorphisms than high-GC regions, and reconciliate discordance between mitochondrial and nuclear phylogenies in A. cerana. Our results shed light on the contribution of polymorphisms in low-GC regions to differentiated region formation along the speciation continuum and their application in reconstruction of intraspecific phylogeny.