The tests were carried out in Murcia, Asturias, Castile and León, and Castile–La Mancha, Spain's Agriculture Minister told Spain's Congress on Monday.
They showed that more than four percent of the beef tested from those zones actually contained DNA from horses, reported El Periódico on Tuesday.
Speaking in congress, Agriculture Minister Miguel Arias Cañete said his ministry and the Ministry of Health had carried out a total of 189 tests to obtain the result.
The tests come after the European Commission previously asked countries to check levels of horsemeat found in products that were labelled as being other types of meat.
Among the 116 tests done by the Agriculture Ministry, six came up positive for meat from horses.
But Cañete hailed what he considered a low result and said this was a sign detection mechanisms were working well.
He said Spain had good testing mechanisms and that any problems in the food chain would quickly be resolved.
The Agriculture Minister also pointed out that no phenylbutazone had been found in any of the samples tested.
The European Commission had also asked Spain to check for traces of the pain reliever for animals which is banned in all products for human consumption.
The Spanish government is now also looking at revising the penalties in its food law to see if these are strict enough.