Here we extend the previous reference alignment sets significantly in terms of the number and diversity of alignments and the number of sequences per alignment. We present an updated benchmark on the formerly identified "good aligners" and (fast) sequence alignment programs using new or optimized program versions. The performance of programs is rated by Friedman rank sum and Wilcoxon tests. We restricted our selection of alignment programs to multiple "sequence" alignment programs because – at least for the computing resources available to us – most structural alignment programs are either too time and memory demanding, or they are restricted to pairwise alignment. Next, we demonstrate for several programs that default program parameters are not optimal for RNA alignment, but can easily be optimized. Furthermore, we evaluate the influence of sequence number per alignment on program performance. One major conclusion is that iterative alignment programs clearly outperform progressive alignment programs, particularly when sequence identity is low and more than five sequences are aligned.