BackgroundPullulanases are the significant industrial group in the 13 glycosyl hydrolases category, known as the α-amylases family. There are very few reports on pullulanase from fungal sources. Based on the above research gap, the present study was undertaken to explore the endophytic fungi for their pullulanase-producing capabilities. ResultsA total of 126 endophytes were isolated from Tradescantia pallida, Zea mays, and Trifolium alexandrinum. Aspergillus, Penicillium, and Ganoderma species recovered highest from the stem of Tradescantia palida. Fusarium was dominant in the stem and leaf of Zea mays. Penicillium, Aspergillus, Ganoderma, Cladosporium, Fusarium, and Alternaria were recovered from the Trifolium alexandrium. The Shannon index in Tradescantia pallida was highest in leaves while in Zea mays and Trifolium alexandrinum, it is highest in the stem. The Simpson’s index is highest in the case of Zea mays stem and root. Species richness was indicated by Menhinick’s index, and it was found that this value was highest in the roots of Trifolium alexandrinum. As per our knowledge, no comparative data is available on the endophytic diversity of the above plants taken for the study. Out of 126 endophytes, only 2.38% produced pullulanase while 7.94% produced amylase. The recovery of pullulanase-producing endophytic fungi was very less. But the importance of pullulanase is high as compared to amylase because it has both α-1,6 and α-1,4 hydrolyzing ability. Therefore, the most promising isolates were identified by ITS sequence analysis. Based on spore chain morphology, isolates BHU-25 and BHU-30 were identified as Penicillium sp. and Aspergillus species, respectively. This is the first report of pullulanase from endophytic Aspergillus and Penicillium. ConclusionEndophytes Aspergillus sp. and Penicillium sp. produce pullulanase enzyme. This is the first report of pullulanase from endophytic Aspergillus and Penicillium.