The study has been initiated with an aim to utilize cytoplasmic male sterile (CMS) associated gene fragment marker to understand the marker flow in segregating population and nature of dominance of the marker. And further it was aimed to understand the best pollination time for the maximum fruit set and to economise the chilli hybrid seed production based on CMS lines. Hence, two CMS based high yielding hybrids, which were found to be much more potential than that of the non CMS based hybrids, have been identified. The CMS gene was dissected from one of the high yielding hybrid. The marker was successfully amplified in A line, F 1 and F 2 population with a polymerase chain reaction (PCR) product of 600 bp. The seedlings were transplanted at the ratio of 2:1, 2:2 and 3:2 sterile:fertile for natural pollination and 2:1 sterile:fertile for artificial pollination. The percent of fruit set was calculated without emasculation (CMS line) and pollination, and with emasculation and pollination, respectively. The maximum fruit set of 95.24% per plant was recorded when artificial crossing attempted between 10:00 am and 11:00 am using male sterile lines. While, 40% fruit set was observed in emasculation and pollination system. The maximum numbers of fruit set (351 and 75) were registered in JNA1 and ACA1 male sterile lines, respectively, thorough artificial pollination. However, the maximum numbers of fruit set (20.24 and 14.74) were recorded in JNA1 and ACA1, respectively, by natural pollination. Pollinating more number of flowers and fruit set success was recorded using male sterile lines than that of the bisexual plant in chilli.