Background: Acute limb ischaemia (ALI) is a limb and life-threatening condition with significant morbidity. There are currently no consensus recommendations for the investigative practices to determine the aetiology of ALI presenting without a known aetiology. We undertook a detailed analysis of all investigations performed to identify an underlying precipitant in those with unexplained ALI and formulated a suggested diagnostic algorithm for the evaluation of unexplained ALI. Methods: ALI cases presenting to a tertiary referral centre over a 3-year period were reviewed, and known aetiologies, and investigations undertaken to determine the underlying aetiology of unexplained ALI were obtained. Results: Unexplained ALI was found in 27 of 222 patients (12%), of which 21 (78%) had a cause for ALI established after further investigations. Six patients had no cause identified despite extensive work-up. Most patients with unexplained ALI had a cardioembolic source identified as the underlying cause (62%), and this included atrial fibrillation, infective endocarditis, cardiac myxoma and intra-cardiac thrombus. Other causes of unexplained ALI were detected by computed tomography (CT) imaging and included newly diagnosed significant atherosclerotic disease (19%), embolism from isolated proximal large vessel thrombus (10%) and metastatic malignancy (10%). There were no cases attributed to inherited thrombophilias, myeloproliferative neoplasms or anti-phospholipid syndrome. Conclusion: Among patients with unexplained ALI, the majority had a cardioembolic source highlighting the importance of comprehensive cardiac investigations. A subset of patients had alternative causes identified on CT imaging. These data support the use of a collaborative and integrative diagnostic algorithm in the evaluation of unexplained ALI.