Abstract Background Acute kidney injury (AKI) is a frequent and clinically relevant problem in critically ill patients. Various randomized controlled trials (RCT) have attempted to assess potentially beneficial treatments for AKI. Different approaches to applying the Kidney Disease Improving Global Outcomes (KDIGO) criteria for AKI make a comparison of studies difficult. The objective of this study was to assess how different approaches may impact estimates of AKI incidence and whether the association between AKI and 90-day mortality varied by the approach used. Methods Consecutive acutely admitted adult intensive care patients were included in a prospective observational study. AKI was determined following the KDIGO criteria during the first 7 days of ICU admission. In this post hoc analysis, we assessed whether AKI incidence differed when applying the KDIGO criteria in 30 different possible methods, varying in (A) serum creatinine (sCr), (B) urine output (UO), and (C) the method of combining these two into an outcome, e.g., severe AKI. We assessed point estimates and 95% confidence intervals for each incidence. Univariable regression was used to assess the associations between AKI and 90-day mortality. Results A total of 1010 patients were included. Baseline creatinine was available in 449 (44%) patients. The incidence of any AKI ranged from 28% (95%CI 25–31%) to 75% (95%CI 72–77%) depending on the approach used. Methods to estimate missing baseline sCr caused a variation in AKI incidence up to 15%. Different methods of handling UO caused a variation of up to 35%. At 90 days, 263 patients (26%) had died, and all 30 variations were associated with 90-day mortality. Conclusions In this cohort of critically ill patients, AKI incidence varied from 28 to 75%, depending on the method used of applying the KDIGO criteria. A tighter adherence to KDIGO definitions is warranted to decrease the heterogeneity of AKI and increase the comparability of future studies.