Abstract Jasmonates regulate plant development and defence. In angiosperms, the canonical bioactive jasmonate is jasmonoyl‐isoleucine (JA‐Ile), which is formed in Arabidopsis thaliana by JAR1 and GH3.10. In contrast to other jasmonate biosynthesis or perception mutants, however, gh3.10 jar1 knockout lines are still fertile. Therefore we investigated whether further jasmonates and GH3 enzymes contribute to regulation of fertility. Jasmonate levels were analysed by liquid chromatography–mass spectrometry. The substrate range of recombinant GH3.10 and related GH3 enzymes was studied using non‐targeted ex vivo metabolomics with flower and leaf extracts of A. thaliana and in vitro enzyme assays. Jasmonate application experiments were performed to study their potential bioactivity. In flowers and wounded leaves of gh3.10 jar1 knockout lines JA‐Ile was below the detection limit. While 12‐hydroxy‐JA was identified as the preferred substrate of GH3.10, no other recombinant GH3 enzymes tested were capable of JA‐Ile formation. Additional JA conjugates found in wounded leaves (JA‐Gln) or formed in flowers upon MeJA treatment in the absence of JA‐Ile (JA‐Gln, JA‐Asn, JA‐Glu) were identified. The aos gh3.10 jar1 was introduced as a novel tool to test for the bioactivity of JA‐Gln to regulate fertility. This study found JAR1 and GH3.10 are the only contributors to JA‐Ile biosynthesis in Arabidopsis and identified a number of JA conjugates as potential bioactive jasmonates acting in the absence of JA‐Ile. However, their contribution in regulating fertility is yet to be conclusively determined.