Tympanic membrane (TM) temperature asymmetry has been proposed as a phenotypic marker of vulnerability to negative emotionality in children. Little is known about the stability of TM temperatures or how readily one can obtain a reliable index of the phenotype. TM temperatures were collected from 3- to 5-year-old children (N = 73) over 5 months (Mdn = 34 samples). Seven to 10 samples needed to be aggregated to achieve reliable measures (αs > .70). Using reliable estimates, TM asymmetry remained stable over time, r(72) = .72, p < .01; 70% of the children had warmer left TMs; and TM asymmetry correlated as predicted with measures of children's emotionality. TM asymmetry may be a phenotypic marker of vulnerability to negative emotionality, but measures obtained on one occasion may not reliably index the phenotype.