Line x tester analysis using four lines as females and eight testers as males was carried out with a view to estimate heterosis in cotton. The experimental material comprised of twelve parents and their thirty two crosses and two standard checks G. Cot. Hy.10 and G. Cot.Hy. 12. A complete set of forty six genotypes was planted in Randomized block design in three replications at the Main Cotton Research Station, NAU, Surat during kharif 2014-15. In case of parents GSHV 171, NDLH 1928 and GISV 267 exhibited higher seed cotton yield per plant. Significant and desired directional heterosis was observed for majority of the yield attributing characters. For seed cotton yield per plant, the higher heterobeltiosis as well as the highest standard heterosis over both the standard checks was recorded by GISV 267 x NDLH 1928. High heterosis for seed cotton yield per plant in this hybrid was due to positive heterotic response in other yield related characters. Combining ability analysis revealed significant and positive variances due to general combining ability (?2GCA) for all characters except number of sympodia per plant which revealed the importance of additive gene action in inheritance of these characters. Similarly, specific combining ability (?2SCA) variances were observed significant for all the characters except lint index and 2.5% span length showed the role of non-additive gene action in inheritance of these traits. The ratio of ?2GCA / ?2SCA revealed that majority of the characters manifested less than unity which indicated preponderance of non-additive genetic variance for inheritance of these traits.