Background Sputum microscopy has remained the cornerstone for tuberculosis diagnosis in low and middle income countries but a large number of sputum samples submitted for microscopic examination is of insufficient quality. Sputum collection is the first and important step for AFB to be detected microscopically. Overnight collection method of sputum was introduced to improve specimen quality and hence increase microbiological yield. Objective To compare the sputum microbiological yield between overnight pooled and standard spot-morning collection method in PTB suspected patients attending KNTH, Kilimanjaro region, Tanzania Materials and Methods PTB Suspected patients who were admitted at KNTH were included in the study. Three (spot-morning and overnight pooled) sputum specimens were collected from each patient. Blood was taken for CD4+T lymphocytes and CXR were taken. Sputum specimens were processed at KCMC microbiology for quality assessment, ZN smear, cultured in MGIT, 7H11 and LJ media. Data were analyzed using STATA computer software version 11 and summarized using tables Results 150 sputum specimen were collected from 50 patients.46 (92%) of patients had overnight pooled specimen of good volume, larger ROC area and early time to positivity in MGIT. There was no difference in sputum thickness, presence of blood and color, AFB smear density, area and CFU count in Middlebrook medium (7H11) between standard and overnight pooled collection method. There was a correlation of overnight pooled sputum CFU count with cavities and CD4+T lymphocytes among HIV negative while no correlation was seen with patient’s HIV status or CD4+ count in HIV positive patients Conclusion Overnight pooled sputum has a good quality of sputum, high sensitivity by having large ROC area and reduced time to positivity in MGIT but no difference in AFB smear density and CFU count with standard method. Correlation of overnight pooled was observed with CD4 in HIV negative and CXR with cavities but none with HIV positive However a study to patients with early signs and symptoms of PTB is recommended before generalization of the results