In this study, seismic fragility analysis of multi-span steel truss bridges still giving service in railway network of Turkey was aimed. For this purpose, two representative multi-span steel truss bridges were selected in Turkish railway lines. A detailed three-dimensional inelastic finite element (FE) model for each bridge was created using commercial FE software. These models were used to perform non-linear time history analyses for each of the selected 30 ground motions scaled to 10 different peak ground acceleration (PGA) values to assess seismic demands of the bridges. To develop fragility curves, probabilistic seismic demand models (PSDMs) were defined. The most appropriate ground motion intensity measure (IM) for PSDMs of both bridges was determined among 11 IMs and the PGA was found as the optimal IM. Finally, the fragility curves for both the components and structural systems of the bridges were developed. The results clearly showed that the multi-span continuous (MSC) steel truss bridge is more vulnerable than the multi-span simply supported (MSSS) steel truss bridge, and for both bridge types, steel bearings are the most vulnerable components. Additionally, top wind braces for the MSC bridge as well as transverse beams and truss vertical members for the MSSS bridge were found as the most vulnerable superstructure members.