Smart card fare systems have become a valuable source of information for public transport OriginDestination (O-D) estimation, allowing a better understanding of individual travel patterns and improving strategic public transport planning. The O-D matrix is important for transportation analysis, design, and management. It gives indispensable information on the travel demand between two different locations, which is used in many transportation applications from strategic planning to traffic control and management.High quality traffic information is required to improve the estimation of O-D matrix in public transportation. Many transit agencies around the world are now using smart card systems to replace traditional payment methods as a viable payment option. Furthermore, the smart card improves the quality of the data; increases the amount of statistics available; updates data continuously 24 hours per day; reduces boarding and alighting time and the driver's workload; saves costs in data collection and editing, eliminates human errors; and provides new opportunities for innovative and flexible fare structuring.Most Automated Fare Collection (AFC) systems, especially when implemented on buses, record passenger boarding information; however, no alighting information is recorded. The lack of alighting stop details is the result of the installed AFC systems, where passengers are not required to use their cards when alighting. Given the information limitations of many AFC systems, the accuracy level of the estimated O-D matrices is often unknown. In addition, many of the assumptions made in suchmethods are yet to be tested.Although smart card fare data has attracted consideration attention as a rich and comprehensive source of information, it has some limitations which constrain its application. One of these limitations is the missing information of passengers' trip purpose. The main reason for the missing information is due to its original use being the collection of revenue and not data collection. The smart card data analysed in the current research was obtained from Translink, the public transport authority of South East Queensland (SEQ), Australia. An important aspect of this system is that it includes both boarding and alighting times and locations, where a passenger gets on or off a public transport vehicle.The first stage of the current research deals with implementing, validating and improving the tripchaining method and its assumptions to estimate public transport O-D matrices using smart card fare data. Transfer time threshold, transfer walking distance, and the location of the last destination of a passenger in a given day, are the major assumptions investigated here. The available rich and unique ii smart card fare data allowed the current investigation and validation of the effect of different tripchaining method assumptions on the estimated matrices. The estimated O-D results are validated with the actual matrices, which are based on full boarding and alighting information. The errors distributi...