At present isolates of Hop stunt viroid (HSVd) are divided into five groups: three major groups (plum-type, hop-type and citrus-type) each containing isolates from only a limited number of isolation hosts and two minor groups that were presumed to derive from recombination events between members of the main groups. In this work we present the characterization of 16 new sequence variants of HSVd obtained from four Mediterranean countries (Cyprus, Greece, Morocco and Turkey) where this viroid had not previously been described. Molecular variability comparisons considering the totality of the sequence variants characterized so far revealed that most of the variability is found in the pathogenic and variable domains of the viroid molecule whereas both the terminal right (T(R)) and left (T(L)) domains are regions of low or no variability, respectively, suggesting the existence of constraints limiting the heterogeneity of the sequence variants. Phylogenetic analyses revealed that sequence variants belonging to the two minor recombinant subgroups are more frequent than previously thought. When the cruciform structure alternative to the typical rod-like conformation was considered it was observed that the upper part of this structure (hairpin I) was strictly conserved whereas in the lower part a reduced variability was found. The existence of a covariation in this lower part was notable. Interestingly, a hammerhead-like sequence was found within the T(R) domain of HSVd and it was strictly conserved in all the sequence variants. The evolutionary implications of the presence of this motif on the HSVd are discussed.