This paper presents a recent study completed on assessing fatigue induced damage to stay cables for an in-service 616 m long light rail transit bridge with a main span of 340 m. In addition to numerical investigations, the study carried out field investigations of bridge deck ambient free-vibration and selected stay cable vibration frequency measurements to establish fundamental dynamic characteristics of the in-service bridge and permanent tension forces in the stay cables. The field results were used to calibrate the numerical model to facilitate the fatigue damage assessment of the stay cables. The fatigue assessment required realistic train loading information including vehicle types and train configurations; historic and future train passage data; and passenger traffic counts during peak and off-peak hours to develop a realistic train histogram to calculate the fatigue demands. As the positioning of the simultaneous train loading on the bridge deck is critical to the assessment, a train crossing study was completed to determine the timing and positioning of two bound trains crossing over the bridge deck simultaneously. The study identified a number of stay cables that are highly vulnerable to pre-mature fatigue failure under the current loading conditions. However, the study also demonstrates that fatigue life of the critical stay cables can be extended by optimizing departure time of trains in opposing directions to avoid simultaneous train passage over the bridge deck at the most critical locations. It can be concluded from the study that the fatigue assessment of stay cables is more complicated and imperative for light rail transit bridges than for highway bridges and that stay cables can be vulnerable to pre-mature fatigue failure even if the original design was based on modern bridge design standards.