After appearance of blue mould ( Peronospora tabacina Adam) about 40 years ago, Croatian tobacco breeders introduced the blue mould resistant flue-cured tobacco F₁ hybrid cultivars into commercial tobacco production. About 20 years ago the major problem became PVY and, the PVY resistant F₁ hybrid cultivars have been grown since then. The earliest grown cultivar was H10, followed by H31, DH10, VaDK, DH17, and DH27. In this study agronomic, morphological and chemical traits and smoking quality evaluated by cigarette manufactures, of these cultivars were studied. The experiment was organized as RCBD and was carried out at two locations during three years. The check variety in the experiment was NC55. Genetic improvement of the traits during the last four decades has been determined. Yield and value increased significantly over the period of time. The average price has changed less. The average yield, price and value of the first domestic cultivar H10 were 2305.83 kg ha^-1, 11.34 kn kg^-1* and 28169.16 kn ha^-1 respectively. The last cultivar accepted by farmers, DH27, had average yield 2902.93 kg ha^-1, price 12.94 kn kg^-1 and value 38107.80 kn ha^-1. At the same time the cultivar NC55 had average yield 2789.58 kg ha^-1 price 13.15 kn kg^-1 and value 37366.71 kn ha^-1. Smoking quality was improved too. Genetic diversity among the studied cultivars has been studied by AFLP molecular markers. The tobacco germplasm used for breeding development of studied cultivars was of Australian (resistance to blue mould and PVY), of European (resistance to PVY), and of N. American origin (leaf yield and quality). Results of the molecular analyses showed grouping of the studied cultivars in accordance with their pedigrees.