Acidovorax citrulli causes bacterial fruit blotch of cucurbits (BFB), and can be assigned to two genetically distinct groups: I and II. The natural association of group I and II A. citrulli strains with different cucurbit hosts suggests host preference. Additionally, in field experiments we observed significant differences in BFB incidence between representative group I and II A. citrulli strains on watermelon and melon fruits, but not on foliar tissues. Thus, we hypothesized that A. citrulli host specificity occurs in cucurbit fruit. The objective of this study was to further characterize differences in the abilities of group I and II A. citrulli strains to colonize melon fruit tissues. Group I and II A. citrulli strains were inoculated on detached melon fruits by syringe-inoculation and attached melon fruits by swab-inoculation. Both group I and II A. citrulli strains colonized melon fruit mesocarp tissues after syringe inoculation and the type 3 secretion system was important for bacterial colonization of internal fruit tissues. However, only group I strains penetrated into pericarps of attached melon fruits after swab-inoculation. These data support the hypothesis that A. citrulli groups display a preference for certain cucurbit fruits and indicate that the mechanisms of host preferential association involve the initial bacterial penetration into the fruit pericarp.