Watermelon seeds can become infested by Acidovorax citrulli, the causal agent of bacterial fruit blotch (BFB) of cucurbits via penetration of the ovary pericarp or by invasion of the pistil. This study investigated the effect of these invasion pathways on A. citrulli localization in seeds. Seed samples (n == 20 or 50 seeds/lot) from pistil- and pericarp-inoculated lots were dissected into testa, perisperm-endosperm (PE) layer, and embryo tissues and tested for A. citrulli by species-specific polymerase chain reaction (PCR) and by plating on semiselective media. Less than 8% of the testa samples were A. citrulli-positive regardless of the method of seed inoculation. Additionally, the difference in percentages of contaminated testae between the two seed lot types was not significant (P == 0.64). The percentage of A. citrulli-positive PE layer samples as determined by real-time PCR assay was significantly greater for seeds from pistil-inoculated lots (97%) than for seeds from pericarp-inoculated lots (80.3%). The mean percentage of A. citrulli-positive embryo samples was significantly greater for seeds from pistil-inoculated lots (94%) than for seeds from pericarp-inoculated lots (approximate to a parts per thousand 8.8%) (P == 0.0001). Removal of PE layers and testae resulted in a significant reduction in BFB seed-to-seedling transmission percentage for seeds from pericarp-inoculated lots (14.8%) relative to those from pistil-inoculated lots (72%). Additionally, using immunofluorescence microscopy, A. citrulli cells were observed in the PE layers and the cotyledons of pistil-inoculated seeds but only in the PE layers of pericarp-inoculated seeds. These results suggest that pericarp invasion results in superficial contamination of the testae and PE layers while pistil invasion results in the deposition of A. citrulli in seed embryos.