Abstract Background Disruption of biological rhythms has been linked to the pathophysiology of mental disorders. However, little is known regarding the rhythmicity of mood symptoms due to the lack of validated clinical questionnaires. A better understanding of the rhythmicity of mood symptoms can help identifying individuals whose severity of mood symptoms follows an altered circadian rhythm. The objective of this study was to validate the English version of the Mood Rhythm Instrument (MRhI), a self-reported measure of self-perceived rhythmicity of mood symptoms and behaviours, in a sample of the general population from Canada. Methods After the translation process, the final English version of the Mood Rhythm Instrument (MRhI-English) was applied on participants recruited at McMaster University and St. Joseph’s Healthcare Hamilton campuses. Individuals were also asked to answer the Reduced Morningness-Eveningness Questionnaire (rMEQ). Results Four hundred one individuals completed the English version of the MRhI and the rMEQ. The MRhI-English presented a Cronbach’s alpha of 0.75. The factorial analysis grouped the MRhI-15 items in 3 factors (cognitive, affective and somatic), with affective items having a lower frequency of self-reported 24-h peaks. Comparison between sexes showed that women reported a higher frequency of daily peaks in irritability, anxiety, sadness and talking to friends, while men exhibited peaks more frequently in problem-solving, sexual arousal and motivation to exercise. Conclusions Our findings suggest that the English version of the MRhI displayed good internal consistency. Future directions will include the use of the MRhI instrument in individuals with mood disorders, aiming to provide a better understanding of the relationship between daily patterns of mood variability and mental health outcomes.