In the present work a total of 228 pooled nasal swabs, 228 pooled tracheal swabs samples from the ailing birds and 152 pooled tracheal tissues, 152 pooled lung tissues from the dead birds were collected from the 19 suspected poultry farms in A.P. and were labelled farm wise and specimen wise. The DNA was extracted from the samples by using Trizol method. The PCR and SYBR Green real time PCR tests was standardized by targeting 16 s rRNA gene. The study found that 12 farms were found to be positive for E.coli, with percent positivity of 63.15% by PCR and 71.2% by SYBR Green Real Time PCR. Among the 12 positive farms, 92(40.35% nasal swabs),106(51.75% tracheal swabs),64 (42.10% tracheal tissues) and 72 (47.36% lung tissues) were positive for E.coli by PCR and 118(49.56% nasal swabs),128(56.14% tracheal swabs),68(44.73% tracheal tissues) and 77(50.65% lung tissues) were positive for E.coli by SYBR Green Real Time PCR, nasal swabs and tracheal swabs were positive with positivity of 40.35 and 51.75% respectively by PCR , 106 and 128, nasal swabs and tracheal swabs were positive with positivity 46.49 and 56.14% by SYBR Green Real Time PCR. This study found that the confirmatory diagnosis of respiratory infections in poultry is accurate when histopathology, isolation and molecular detection methods like PCR and SYBR Green Real time PCR are used.