The Life Orientation Test (LOT-R) is the most intensively used instrument for the assessment of optimism, but empirical evidence of its psychometric quality in Latin America is lacking. The aim of this study was to test psychometric properties of this questionnaire based on a representative sample of the general population of Colombia (N = 1,500). Confirmatory factor analyses confirmed the bi-dimensionality of the questionnaire with two factors, namely Optimism and Pessimism. The sum scores of the optimism and pessimism subscale correlated with r = −.12. Convergent validity aspects were studied by correlating LOT-R values with anxiety, depression, quality of life, fatigue, mental health, hopelessness, and self-efficacy. Optimism was more strongly correlated with these variables than pessimism. Normative data and mean values for both genders and different age groups are given. Generally, there were only small age and gender effects. Compared to the German norm data, the Colombian participants scored on average one scale point higher in the dimension of optimism. In summary, the LOT-R in its Spanish version is an appropriate and practical tool for screening purposes in individual diagnostics and epidemiological research in Latin American samples.