The Gratitude Questionnaire is a short, self-report measure of the disposition to experience gratitude. The Gratitude Questionnaire has been validated in several countries but its factor structure remains controversial. Therefore, the main goal of the study was to examine the factor structure of the Gratitude Questionnaire in a Spanish sample. Two samples were recruited (957 and 920 participants). The confirmatory factor analyses showed that the best fit was the five-item model with errors of item four and five correlated (CFI = .99, NFI = .99, RMSEA = .02). This model demonstrated partial cross-validity based on an analysis of factorial invariance. The Composite Reliability of the five-item Gratitude Questionnaire was .81. In addition, it was found that gratitude was positively related to subjective and psychological well-being. Specifically, the Gratitude Questionnaire was positively correlated to life satisfaction (r = .56, p < .01), affect balance (r = .46, p < .01), self-acceptance (r = .54, p < .01), positive relations (r = .44, p < .01), autonomy (r = .17, p < .01), environmental mastery (r = .49, p < .01), personal growth (r = .36, p < .01), and purpose in life (r = .50, p < .01). According to the results, it can be concluded that the Spanish version of the five-item Gratitude Questionnaire possessed better psychometric properties than the original six-item model.