Habitat fragmentation is likely to have deleterious genetic consequences for plant populations. Although the genetic effects of fragmentation in plants have been investigated in various landscapes, such studies are scarce in urban landscapes where forests tend to be fragmented and have a complex internal structure.This study aimed to determine the factors, including patch and sub-patch level spatial factors, affecting the genetic diversity of a herbaceous species in urban fragmented forests.We collected 30-39 leaf samples of Viola grypoceras A. Gray var. grypoceras, a perennial herbaceous species with short-distance seed dispersal, from 12 fragmented and 12 suburban forests each at Kyoto City, Japan, and analyzed the genetic diversity of this species by developing six simple sequence repeat markers. Field survey was conducted to collect demographic and spatial data.There was no significant difference in allelic richness between the urban fragmented and suburban forests. However, statistical analysis revealed that the area of vegetation, distribution pattern of populations in a forest, and average distance between nearest populations affected the genetic diversity of this species in urban fragmented forests.Although V. grypoceras has traits that allow it to tolerate fragmentation, such as self-pollination and seed bank-formation ability, pure loss of habitat and reduced fragment size might have deleterious effects on this species, and these effects might become more apparent if fragmentation continues to proceed in the future.