To study the effect of NAA and Zn-EDTA application on yield and fiber quality of cotton cultivars, an experiment based on split-split plot arrangement with randomized complete block design in three repetitions was conducted in two locations (Varamin and Razeghabad regions) of Tehran province, Iran in 2013–14. In this research, the main plot included three cotton cultivars (Varamin, Khordad and Pak), the sub-plots were sprayed by spraying with pure water (control), Zn-EDTA with 1 ml/l and 2 ml/l concentration and NAA with concentration of 30 ppm and control were as sub-sub plots that were at four levels (control, one time of spraying, two times spraying and three times spraying). The results showed that Zn-EDTA spraying with appropriate concentration alongside using NAA as the spray resulted in increased number of total bolls per plant, increased weight of single bolls, seed cotton yield in 1st and 2nd picking, total seed cotton yield and fiber length compared to the control group. Zn-EDTA spraying did not have a significant effect on fiber uniformity and fiber strength. There was a significant difference between cultivars in terms of fiber length, fineness and strength. However, using NAA did not have significant effect on fiber uniformity, strength and fineness. In total, reaction of different cultivars in two locations to different Zn-EDTA concentrations and NAA sprayed were different in most surveyed characteristics.