Spain has inevitably used the UK’s EU referendum to bolster its argument that it should take back control of Gibraltar, home to many of Europe’s leading e-gaming firms.
The interim Spanish Foreign Minister Jose Manuel Garcia-Margallo stated in an interview that Spain would “be talking about Gibraltar the very next day [after the referendum]” if the UK votes to leave the EU in June.

Gibraltar is well-used to this posturing by its neighbour and a statement from the Gibraltese government said in response: “The declared intent of the caretaker Foreign Minister of Spain to bring the question of Gibraltar to the fore in the event of the UK and Gibraltar leaving the EU confirms the analysis that has already been made. It is safer and more secure for Gibraltar to remain in the EU in order to deny Mr Margallo the opportunity to pounce on us.” 

At present the border between Spain and Gibraltar is not within the EU Schengen Area because the UK itself has opted out of the borderless Schengen Area. There can be delays of several hours to cross the border at peak times.
Spain ceded Gibraltar to the British as part of the Treaty of Utrecht, signed in April 1713 after the War of Spanish Succession.
The betting suggests that the UK will remain in the EU (1/3 with William Hill) rather than vote to leave (9/4). But in the event that the odds are upset in June, you can bet the Spanish will not let the issue of Gibraltar drop.