A crystallographic study and theoretical analysis of the Si/Ga site preferences in the Gd5GaxSi4-x series is presented. Gd5GaxSi4-x adopt the orthorhombic Gd5Si4-type structure (space group Pnma, Z = 4) with a maximum Ga content near x = 1.00, as determined by single crystal and powder X-ray diffraction. Refinements from single crystal X-ray diffraction studies of the three independent sites for Si/Ga atoms in the asymmetric unit (interslab T1, intraslab T2 and T3) reveal partial mixing of these elements, with a clear preference for Ga substitution at the interslab T1 sites. To investigate site preferences of Si/Ga atoms, first-principles electronic structure calculations were carried out using the Vienna ab initio simulation package (VASP) and the Stuttgart tight-binding, linear-muffin-tin orbital program with the atomic sphere approximation (TB-LMTO-ASA). Analysis of various crystal orbital Hamilton population (COHP) curves provide some further insights into the structural tendencies and indicate the roles of both sizes and electronegativities of Ga and Si toward influencing the observed upper limit in Ga content in Gd5GaxSi4-x. The magnetic properties of two Gd5GaxSi4-x phases are also reported: both show ferromagnetic behavior with Curie temperatures lower than that for Gd5Si4.