Animal exchange networks have been shown to play an important role in determining gene flow among domestic animal populations. The Silk Road is one of the oldest continuous exchange networks in human history, yet its effectiveness in facilitating animal exchange across large geographic distances and topographically challenging landscapes has never been explicitly studied. Horses are known to have been traded along the Silk Roads; however, extensive movement of horses in connection with other human activities may have obscured the genetic signature of the Silk Roads. To investigate the role of the Silk Roads in shaping the genetic structure of horses in eastern Eurasia we analysed microsatellite genotyping data from 455 village horses sampled from 17 locations. Using least-cost path methods, we compared the performance of models containing the Silk Roads as corridors for gene flow with models containing single landscape features. We also determined whether the recent isolation of former Soviet Union countries from the rest of Eurasia has affected the genetic structure of our samples. The overall level of genetic differentiation was low, consistent with historically high levels of gene flow across the study region. The spatial genetic structure was characterised by a significant, albeit weak, pattern of isolation by distance across the continent with no evidence for the presence of distinct genetic clusters. Incorporating landscape features considerably improved the fit of the data; however, when we controlled for geographic distance, only the correlation between genetic differentiation and the Silk Roads remained significant, supporting the effectiveness of this ancient trade network in facilitating gene flow across large geographic distances in a topographically complex landscape.