A set of Safety Performance Function (SPFs) commonly known as accident prediction models, were developed for evaluating the safety of Highway segments under the jurisdiction of Ministry of Transportation, Ontario (MTO). A generalized linear modeling approach was used in which negative binomial regression models were delevoped separately for total accidents and for three severity types (Property Damage Only accidents, Fatal and Injury accidents) as a function of traffic volume AADT. The SPFs were calibrated from 100m homogenous segments as well as for variable length continuous segments that are homogeneous with respect to measured traffic and geometric characteristics. For the models calibrated for Rural 2-Lane Kings Highways, the variables that had significant effects on accident occurrence were the terrain, shoulder width and segment lenght. It was observed that the disperson parameter of the negative binomial districution is large for 100m segments and smaller for longer segments. Further investigation of the dispersion parameter for Rural 2-Lane Kings Highways showed that the models calibrated with a separate dispersion parameter for each site depending on the segment length performed better that the model calibrated considering fixed dispersion parameter for all sites. For Rural 2-Lane Kings Highways, a model was calibrated with trend considering each year as a separate observation. The GEE (Generalized Estimating Equation) procedure was use to develop these models since it incorporated the temporal correlation that exists in repeated measurements. Results showed that integration of time trend and temporal correlation in the model improves the model fit.