Background: Gemcitabine and cisplatin (gemcis) has been the standard of care for biliary tract cancer (BTC) for over a decade. One US model predicted the total lifetime cost of gemcis per patient to be $54,077 in 2020 CAD, although not all costs were considered. Our study objectives included quantifying updated costs and health care resource utilization (HCRU) associated with advanced BTC using real-world, population-level data from Ontario databases. Methods: We conducted a retrospective population-level study of patients diagnosed with recurrent or unresectable/advanced BTC (gallbladder cancer [GBC], intrahepatic and extrahepatic cholangiocarcinoma [IHC and EHC], Ampulla of Vater [AoV]) between January 1, 2010 and December 31, 2019. Follow-up data were available until December 31, 2020. HCRU and costs were calculated from the start date of first line (1L) of BTC-specific systemic therapies to death or the end of March 31, 2020 whichever occurred first. A macro-based costing methodology (GETCOST) was used and the mean cost per patient was reported in 2020 Canadian dollars (CAD). Mean cost per patient was calculated from diagnosis till the end of follow-up or death. The total number of HCRU-specific encounters for all years was divided by the number of BTC patients who used that HRCU in order to report the mean number of HCRU encounters (e.g., cancer clinic visits and inpatient hospitalizations). Results: Of 2,142 advanced BTC patients identified, 1,968 were diagnosed with non-AoV cancers (GBC, IHC, EHC or general BTC not otherwise specified) and 174 patients were diagnosed with AoV cancer. The mean cost per patient for all 1L-treated patients was $36,662 ($36,378 for non-AoV compared to $40,092 for AoV patients). By 1L-specific therapies, the highest mean cost per patient was for patients on gemcitabine and taxane combinations (gemtaxane) at $44,190, followed by gemcitabine and cisplatin (gemcis) at $34,507, then FOLFOX or FOLFIRI (fol) at $39,002 and carboplatin and gemcitabine (gemcarb) at $30,905. Untreated patients had a mean cost per patient of $42,136. The mean number of cancer clinic visits per patient per year during 1L ranged from 9.7 (untreated) to 13.7 (gemcis), while the inpatient hospitalizations per patient per year during 1L ranged from and 1.5 (gemcarb, fol and gemtaxane) to 2.4 (untreated). Conclusions: This study reports Canadian-specific, real-world costs and HCRU results of over 2,000 BTC patients. While cost results were stratified by type of BTC (AOV and non-AOV) and type of 1L therapies, the mean cost per patient ranged from $30,905 CAD to $44,190 CAD and HCRU results indicated substantial mean number of cancer clinic visits and inpatient hospitalizations. Thus, BTC patients incur a significant economic burden to the patients and Ontario public payer health system.