In recent years, fentanyl is increasingly detected in overdose cases as well as human performance cases due to the current opioid epidemic. The comparison of fentanyl concentrations in deaths to recreational fentanyl use found in driving under the influence of drugs (DUID) cases is important to show how increased availability effects the historical knowledge of fentanyl. A two-year review of fentanyl cases from Orange County, CA, USA is presented to highlight fentanyl concentrations in these two case types. Any non-urine sample with fentanyl detected above the limit of detection of 0.5 ng/mL was reviewed in DUID and postmortem cases. The mean and median fentanyl concentrations in 199 antemortem blood samples from DUID cases were 14.2 ng/mL and 5.3 ng/mL, respectively, with a range of 0.5–303 ng/mL. In 285 central blood samples from postmortem cases the mean was 29.1 ng/mL, the median was 16.9 ng/mL and the range was 0.6–636 ng/mL. In a total of 58 postmortem peripheral blood samples, the mean, median and range were 14.0 ng/mL, 10.0 ng/mL and 0.9–78.0 ng/mL, respectively. Of the 55 brain samples analyzed, the fentanyl mean, median and range of fentanyl concentrations were 50.0 ng/g, 31.4 ng/g and 1.9–441 ng/g, respectively. Fentanyl concentrations in 16 liver samples had a mean of 82.0 ng/g, a median of 69.4 ng/g and a range of 11.6–226 ng/g. The overlap of fentanyl concentrations from the antemortem and postmortem samples highlights the importance of evaluating case circumstances when providing an interpretation in postmortem cases. Additional information such as age, gender, polydrug use, and postmortem redistribution is presented for the cases included in the study.