Estimating epidemiological cutoff endpoints (ECVs/ECOFFS) may be hindered by the overlap of MICs for mutant and non-mutant strains (harboring or not harboring mutations, respectively). Posaconazole MIC distributions for Aspergillus fumigatus SC were collected from 26 laboratories (Australia, Canada, Europe, India, South/North America, Taiwan) and published studies. Distributions that fulfilled CLSI criteria were pooled and ECVs were estimated. The sensitivity of three ECV analytical techniques (ECOFFinder, NRI, derivatization) to the inclusion of MICs for mutants was examined for three susceptibility testing methods (CLSI, EUCAST, and Etest®). The totals of posaconazole MICs for non-mutant (no known cyp51A mutations) and mutant A. fumigatus isolates were: by CLSI, 2,223 and 274; by EUCAST, 556 and 52; by the Etest®, 1,365 and 29 respectively; 381 Sensititre™ YeastOne™ (SYO) MICs with unknown mutational status were also evaluated. We observed an overlap in posaconazole MICs among non-mutant and cyp51A mutants. At the commonly chosen percentage of the modeled wild-type population (97.5%), almost all ECVs remained the same when the MICs for non-mutant and mutant distributions were merged: ECOFFinder ECVs 0.5 μg/ml (CLSI) and 0.25 μg/ml (EUCAST and Etest®); NRI ECVs: 0.5 μg/ml for all three methods. However, the 95% ECOFFinder CLSI ECV for non-mutants was 0.25 μg/ml. The tentative SYO ECOFFinder ECV was 0.06 μg/ml (data from 3/8 laboratories). Derivatization ECVs with or without mutant inclusion were either 0.25 μg/ml (CLSI, EUCAST, Etest) or 0.06 μg/ml (SYO). It appears that ECV analytical techniques may not be vulnerable to overlap between presumptive wild-type and cyp51A mutants when up to 11.6% of the estimated wild-type population includes mutants.