The selective deposition and self-assembly of nanospheres from a colloidal suspension in trenches on silicon surfaces is investigated using conventional light, confocal laser scanning and scanning electron microscopy. Trenches with widths of one to several nanosphere diameters are formed on silicon surfaces by photolithography and reactive ion etching. The spreading knife convective self-assembly technique is employed to distribute the nanosphere suspension on the pre-patterned surface. It is shown that this technique is particularly useful in combination with a functionalized surface where a self-assembled molecular monolayer changes the contact angle such that sphere deposition takes place almost exclusively in the trenches. By this, lines selectively filled with a chain of beads with a length of 0.5 mm have been achieved.