Over the last 20 years there has been a significant increase in fentanyl related deaths in Ontario, Canada. This report examines toxicological findings in a series of death investigations in which fentanyl was quantitated to identify the prevalence, trends and demographic data associated with fentanyl in Ontario, Canada, and to highlight the changes in these trends since fentanyl began appearing in casework in Ontario in the early 2000s. A retrospective study of all cases in which fentanyl was quantitated in blood, using liquid chromatography–tandem mass spectrometry, was conducted for the time period between January 1, 2020, and December 31, 2022. A total of 4395 cases were included, 77% of the decedents were male and 23% were female with ages ranging from 0 to 95. The most frequently classified cause of death was mixed drug toxicity (69%) followed by fentanyl intoxication at 19%. Less than 10% of cases where fentanyl was quantitated were classified as non-drug related deaths. Fentanyl concentrations in all cases ranged from 1.3 to > 2000 ng/mL. Other drugs were frequently detected with fentanyl. In mixed drug toxicity cases, stimulants were the most frequently encountered class of drugs: cocaine was identified in 51.8% and methamphetamine was observed in 43.0% of cases, respectively. Detailed reports for select cases were included to provide additional insight into the different case types and to show the difficulty in interpreting blood concentrations without additional detailed case histories. This study provides valuable information for the scientific and medical community regarding the continued use of fentanyl and how patterns of fentanyl use have evolved since it began to appear in forensic casework.