Allopatric speciation may be the principal mechanism generating new species. Yet, it remains difficult to judge the generality of this process because few studies have provided evidence that geographic isolation has triggered the development of reproductive isolation over multiple species of a regional fauna. Here, we first combine results from new empirical data sets (7 taxa) and published literature (9 taxa) to show that the eastern Great Lakes drainage represents a multispecies suture zone for glacial lineages of freshwater fishes with variable levels of genetic divergence. Second, we performed amplified fragment length polymorphism analyses among four pairs of lineages. Results indicate that lineages with relatively deep levels of mtDNA 5′ COI (barcode) sequence divergence (>2%) developed strong reproductive barriers, while lineages with lower levels of divergence show weaker reproductive isolation when found in sympatry. This suggests that a threshold of 2% sequence divergence at mtDNA could be used as a first step to flag cryptic species in North American freshwater fishes. By describing different levels of divergence and reproductive isolation in different co-occurring fishes, we offer strong evidence that allopatric speciation has contributed significantly to the diversification of north-eastern American freshwater fishes and confirm that Pleistocene glacial cycles can be viewed as a ‘speciation pump’ that played a predominant role in generating biodiversity.