Introduction: Various types of separators have been advocated, but the ideal separator should produce optimum separation with minimal pain and discomfort. Objective: The objective of this study was to evaluate and compare the amount of separation achieved by three different types of separators (Elastomeric, Kesling and Kansal), and to assess the associated pain and discomfort. Methods: A random single-blind split-mouth study was conducted on 108 patients seeking fixed orthodontic treatment, in which two different separators were used on each side in both the arches for a single patient. After five days, the amount of separation was measured with a feeler gauge. Visual Analogue Scale (VAS) scoring was performed by the patient on each day, to evaluate pain perception. Discomfort was evaluated by questionnaire filled by the patient at the time of separator removal. Results: The greatest amount of separation was seen with the elastomeric separators, while the smallest separation was seen with Kansal separators. VAS scoring showed maximum pain at day 1 with all the three separator types. Highest pain was perceived in the Elastomeric separators group, followed by Kesling and Kansal separators, respectively. Statistically significant difference was found in VAS score of Elastomeric separators, when compared to both Kesling and Kansal, on day 1 and 2 (p= 0.001). Analysis of the questionnaires revealed that a greater number of patients experienced discomfort with elastomeric separators placement (69.4%), which was statistically significant (p< 0.01) when compared to the other two types of separators. Answers to the other questions were comparable, except for the need for medications, which was reportedly highest with elastomeric separators. Conclusion: Kesling separators produce adequate separation with minimal discomfort and pain, compared to Elastomeric and Kansal separators.