The choice of cortisol sampling times in early childhood studies varies widely. Given that recommendations on sampling protocols are largely based on adults, the present study aimed to broaden current knowledge by examining how reliably cortisol measures obtained at different daytimes would reveal between-individual differences in toddlers' cortisol levels. Parents were instructed to take 10 saliva samples consecutively (five per day) from their toddler (N = 19; M[subscript age] = 15.8 months, SD[subscript age] = 4.2 months). Intra-class correlations (ICCs) were computed to evaluate cortisol reliability. Cortisol samples taken in the morning between 30 and 80 min after awakening and bedtime samples were most reliable in differentiating between children (ICCs [greater than or equal to] 0.80). Wake-up cortisol samples taken within the first 30 min after awakening and afternoon samples showed moderate reliabilities (ICCs = 0.64), whereas the reliability of noon samples was poor (ICC = 0.43). Therefore, when investigating cortisol in young children while being restricted to a few samples only, assessing cortisol in the morning (at least 30 min after awakening) and at bedtime would be advisable.