--- - |2 Summary Outbred rodent stocks including Sprague Dawley rats, are known for their genetic diversity and so they are often used to develop animal models of human disease. Although between‐colony differences in pharmaco‐behavioural studies have been published previously, a direct head‐to‐head comparison study, whereby all research was performed in the same laboratory by the same experimenter utilising the supraspinal route of drug administration in the same strain of rat, is lacking. Herein, we report our head‐to‐head comparison study, involving assessment of antinociception, constipation and respiratory depression evoked by single bolus intracerebroventricular (ICV) doses of morphine, buprenorphine, DPDPE and U69,593 using male Sprague Dawley rats sourced from a different breeding colony (BC2) from that (BC1) used by us previously. Our data show that there are marked differences in the potency rank order for morphine and buprenorphine between rats sourced from BC2 and BC1. Although ICV morphine evoked a bell‐shaped dose‐response curve in the constipation test for rats from both colonies, this occurred at higher doses for rats from BC2. In conclusion, our head‐to‐head comparison shows considerable between‐colony differences for the same rat strain, in the potency rank order of two clinically important strong opioid analgesics given by the ICV route. - Clinical and Experimental Pharmacology and Physiology, Volume 45, Issue 10, Page 1056-1066, October 2018.