The abuse and overdose of opioid drugs are growing public health problems, globally. While progress has been made towards understanding the mechanisms governing tolerance to opioids, the exact cellular machinery involved remains unclear. However, there is growing evidence to suggest that c-Jun N-terminal Kinases (JNKs) play a major role in mu opioid receptor regulation and morphine tolerance. In this study, we aimed to determine the potential roles of different JNK isoforms in development of tolerance to the anti-nociceptive and hypothermic effects of morphine. We used the hotplate and tail-flick tests for thermal pain to measure tolerance to the anti-nociceptive effects of once daily sub-cutaneous injections with 10 mg/kg morphine. Body temperature was also measured to determine tolerance to the hypothermic effects of morphine. Tolerance to morphine was assessed in wild-type mice and compared to single knockout (KO) mice each lacking of the JNK isoforms (JNK1, JNK2, or JNK3). We found that loss of each individual JNK isoform causes impairment in tolerance for the anti-nociceptive and hypothermic effects of daily morphine. However, disruption of JNK2 seems to have the most profound effect on morphine tolerance. These results demonstrate a clear role for JNK signaling pathways in morphine tolerance. This compliments previous studies suggesting that the JNK2 isoform is required for morphine tolerance, but additionally presents novel data suggesting that additional JNK isoforms also contribute to this process.