This repository contains (12×500=)6,000 pairs of nucleotide sequences that have been simulated for testing alignment-free genome distance estimates, as described in Criscuolo (2019). Given an evolutionary distance d varying from 0.05 to 0.60 (step = 0.05), the program SeqGen was used to simulate the evolution of 500 nucleotide sequence pairs with d substitution events per character (GTR+Γ evolutionary model). For each of the 12 evolutionary distances d = 0.05, 0.10, ..., 0.60, an XZ-compressed file containing 500 lines is available. Each line contains 18 fields separated by blank spaces: [1] seed value used during simulation, [2] true evolutionary distance d between the two simulated sequences, [3] total number of simulated characters, [4] number of non-indel characters with nucleotide mismatch, [5] number of non-indel characters, [6-9] A, C, G, T frequencies used during simulation, [10-15] GTR parameters used during simulation, [16] Γ distribution parameter used during simulation, [17-18] two simulated sequences with indel events as gaps. Of note, each pair of aligned sequences without gaps can be regenerated using SeqGen v1.3.4 with parameters from fields [1,3,6-16] and the following two-leaf model tree: (t1:d,t2:0.000); where d is given in field [2]. ___ Criscuolo A (2019) A fast alignment-free bioinformatics procedure to infer accurate distance-based phylogenetic trees from genome assemblies. Research Ideas and Outcomes, 5:e36178. doi:10.3897/rio.5.e36178