The root-deck crack is one of the most concerned fatigue issues on orthotropic steel decks (OSDs) as it is unfavorable for the serviceability of the bridge while being difficult to detect. In this paper, the fatigue test was designed and carried out by using beach marking method to record the crack propagation process. By conducting microscopic examination on the fracture surface, it shows that the lack of penetration (LOP) at the weld root, combined with the high stress at that site, results in the fatigue crack initiation and propagation to penetrate the deck. Additionally, the fatigue assessment in terms of the S-N curves for such a failure mode was carried out. With comparison to other fatigue test results from the literature, it manifests the differences between the specimens failed due to root-deck crack and toe-deck crack, which can benefit the fatigue design of OSDs by giving a more specific and process-oriented results.