This book constitutes the refereed proceedings of the 16th European Conference on Evolutionary Computation in Combinatorial Optimization, EvoCOP 2016, held in Porto, Portugal, in March/April 2016, co-located with the Evo*2015 events EuroGP, EvoMUSART and EvoApplications. The 17 revised full papers presented were carefully reviewed and selected from 44 submissions. The papers cover methodology, applications and theoretical studies. The methods included evolutionary and memetic algorithms, variable neighborhood search, particle swarm optimization, hyperheuristics, mat-heuristic and other adaptive approaches. Applications included both traditional domains, such as graph coloring, vehicle routing, the longest common subsequence problem, the quadratic assignment problem; and new(er) domains such as the traveling thief problem, web service location, and finding short addition chains. The theoretical studies involved fitness landscape analysis, local search and recombination operator analysis, and the big valley search space hypothesis. The consideration of multiple objectives, dynamic and noisy environments was also present in a number of articles.