Yes. It is one of the fundamental things about this game. You want offensive ninjas to be in the first two positions and those with more support/defensive in the 3 and 4.

Wait a minute, what exactly are you asking? Positioning of teams? Like in Ranked Battle?

Like in rows for battles you organize them in the spaces

Of course it matters, it determines in what order your ninjas will attack. And once you unlock the tactics system it will also determine what stat they gain dependant on which slot you put them in.