Introduction. Studies in Ethiopia have indicated that tuberculosis (TB) patient's elapsed a long time before initiating treatment.Gap Statement. However, there is very limited evidence on the association of treatment initiation delay with drug resistance.Research Aim. To investigate the association of delayed treatment initiation with drug resistance among newly diagnosed TB patients in Tigray, Ethiopia.Methods. We conducted a follow-up study from October 2018 to June 2020 by recruiting 875 pulmonary tuberculosis (PTB) patients from 21 randomly selected health facilities. Delays to initiate treatment and drug resistance were collected using a standardized questionnaire and standard laboratory investigation. The association of delay to initiate treatment with acquired drug resistance was modelled using penalized maximum-likelihood (PML) regression models. Data were analysed using stata software version 15. Statistical significance was reported whenever the P-value was less than 0.05.Result. The median total delay to treatment initiation was 62 days with an inter-quartile range of 16-221 days. A unit change in time to initiate treatment reduced the risk of acquired drug resistance by 3 %. Being smear-positive at the end of treatment and after 2 months of treatment initiation were significantly associated with a higher risk of acquired drug resistance. Whereas, having a mild clinical condition was associated with a lower risk of drug resistance.Conclusion. Time to treatment initiation delay is associated with an increased risk of the emergence of drug resistance. Efforts targeted towards reducing the negative effects of PTB should focus on reducing the length of delay to initiate treatment.