Isothermal inactivation experiments often are used to investigate the thermal resistance of pathogens, such as Salmonella, in foods; however, little is known about the reproducibility of such experimental methodologies. The objective of this study was to quantify the reproducibility of Salmonella isothermal resistance results via a six-laboratory comparison. Inoculation was performed at a single location and then distributed to each laboratory for isothermal analysis. Salmonella Agona 447967 was inoculated into oat flour, re-equilibrated to a water activity (aw) of 0.45, and then packaged and distributed to each laboratory. Before conducting the inactivation trials, each laboratory was required to verify the inoculated product's aw, enumerate Salmonella population levels, and verify that the isothermal treatment medium was at the target temperature (80°C). All laboratories were required to process at least three replications, collect at least six sample time points with three subsamples at each sampling point, enumerate survivors using an identical plating methodology and media, and verify that the temperature did not substantially change during isothermal treatment. The log-linear model was fit to the Salmonella survivor data, and the resultant D-values were statistically compared via Welch's t test (α = 0.05). Two significant differences in thermal inactivation kinetics were identified as potentially resulting from suspected methodology deviations. Two of the inoculated batches distributed for analysis yielded significantly lower D-values, which likely resulted from a deviation in the inoculation procedures. One laboratory yielded significantly lower D-values, which was likely the result of temperature deviations. Overall, excluding the D-values resulting from deviations, the inactivation results were reproducible, yielding D-values of 30.2 ± 3 min. These results indicate that isothermal inactivation results can be reproducible but that even minor methodology deviations can substantially affect measured Salmonella thermal resistance.