Background: Human Adenovirus species D (HAdV-D) was common human viral pathogen especially in eye infection, consists of several types of which HAdV-D8,-D19 and –, D37 were common in eye infection. This study includes detection of HAdV-D types implicated in conjunctivitis based on L2 (Penton protein) gene similarity. Methods: Conjunctival swabs were collected from Keratoconjunctivitis patients as eye infection related to adenovirus. Viral nucleic acids were extracted and specific primer pairs for HAdV-D L2 gene (encoding for penton base protein) was used to amplify the target gene and only positive samples were sent to sequencing. Results: The results revealed that only 6 samples give positive results for L2 gene amplification and then sent for sequencing for L2 (penton protein) gene-based typing. The results show that 4 local isolates (S1, S2, S3, S6) were similar to HAdV-D8 and 2 local isolates (S4, S5) were similar to HAdVD20. Also the results display that the HAdV-37, prominent HAdV-D type of human eye infection, may be variant of HAdV-D20 due to that six variation were seen in S4and S5 local isolates nucleotide sequence in relation to HAdV-D37: T>C at position 14364, A>C at position 14411, T>C at position 14427, C>A at position 14448, G>A at position 14540 and T>C at position 14617, leading to only 2 amino acid change in resulted penton protein: T (Threonine) instead of K (Lysine) at position 204 and N (Asparagine) instead of D (Aspartic acid) at position 247. Conclusions: The current study concludes the possibility of implication of HAdV-D20 in eye infections especially conjunctivitis.