Background: Difficulty in cannulation of arteriovenous fistula (AVF) can lead to inadequate dialysis, transient to permanent loss of access and increases dependency on bridging catheters. This study aimed to analyze the causes for difficult fistula cannulation, using various imaging modalities. Methodology: This was a retrospective single-center observational study conducted between October 2017 and June 2018. Patients whose fistulae were difficult to cannulate were initially evaluated by physical examination followed by doppler ultrasonography or/and fistulogram as necessary. The patients were divided into two groups that is, primary difficult cannulation (within first three months of creation of fistula) or secondary difficult cannulation (after three months). Results: We encountered difficult cannulation in 43 patients. About 60% were primary difficult cannulations. Most common causes for difficulty in cannulation were cannulation zone (CZ) stenosis (23.3%), immature fistula (20.9%), outflow stenosis (18.6%), inflow stenosis (11.6%), anatomical abnormalities (11.6%), outflow plus CZ stenosis (9.3%) and inflow plus CZ stenosis (4.7%). Among patients with primary difficult cannulation, immature fistula (34.6%) was the most common cause, whereas CZ stenosis (47.1%) was the most common etiology for secondary difficult cannulation. Edema leading to difficult cannulation was found in 12 patients (27.9%), all of which was due to central vein stenosis. Cannulation resulted in hematoma, fistula thrombosis, failure of fistula and pseudoaneurysm in 83.7%, 27.9%, 16.3%, and 2.3% of cases respectively. Bridging temporary dialysis catheter placement was required in 67.4% patients. Ultrasound doppler had lower diagnostic value when compared to fistulogram (71.4% vs 93.9%, p = 0.014). Conclusion: Difficulty in cannulating the arteriovenous fistula is a common problem in hemodialysis patients. We suggest that patients whose fistulae are difficult to cannulate should undergo early radiological evaluation to decrease catheter dependency and access failure.